Napi2b-targeted antibody-drug conjugates and methods of use thereof

ABSTRACT

This disclosure provides NaPi2b-targeted antibody-drug conjugates (e.g., NaPi2b-targeted antibody-polymer-drug conjugates) that specifically bind to the extracellular region of SLC34A2, and to methods of using such conjugates in a variety of therapeutic, diagnostic, and prophylactic indications.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/457,574, filed Mar. 13, 2017, which claims priority to, and thebenefit of, U.S. provisional application Nos. 62/308,567, filed Mar. 15,2016; 62/323,068 filed Apr. 15, 2016 and 62/383,324 filed Sep. 2, 2016,under 35 USC § 119(e). The contents of each of these applications arehereby incorporated by reference in their entireties.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the file named “MRSN-016_C01US_ST25.txt”, which wascreated on Jan. 20, 2021, and is 30.2 KB in size are hereby incorporatedby reference in their entirety.

FIELD OF THE DISCLOSURE

This disclosure relates generally to NaPi2b-targeted antibody-drugconjugates (such as NaPi2b-targeted antibody-polymer-drug conjugates)that specifically bind to the extracellular region of SLC34A2, and tomethods of using these conjugates as therapeutics and/or diagnostics.

BACKGROUND

NaPi2b (SLC34A2, NaPiIIb, Npt2), a multi-transmembrane, sodium-dependentphosphate transporter (Xu et al. Genomics 62:281-284 (1999)), isnormally expressed at the brush border membrane of mammalian smallintestine and participates in the transcellular inorganic phosphate (Pi)absorption, contributing to the maintenance of phosphate homeostasis inthe body. The expression of NaPi2b at the protein level has beendetected in the liver, at the apical surface of epithelial cells ofmammary, salivary glands, and in the lungs, testis, salivary gland,thyroid gland, small intestine, and uterus. Mutations in NaPi2b havebeen associated with clinical syndromes of alveolar and testicularmicrolithiasis. NaPi2b is highly expressed in non-squamous non-smallcell lung cancer (NSCLC), non-mucinous ovarian cancer and papillarythyroid cancer. NaPi2b-positive tissue immunoreactivity is present in61% of NSCLC, and 92% ovarian cancer specimens.

Ovarian cancer is one of the most common gynecologic malignancies andthe fifth most frequent cause of cancer death in women. The highmortality rate results in part from the frequent diagnosis of ovariancancer at advanced stages and the mortality rate is approximately 65% ofthe incidence rate. Epithelial tumors of ovary comprise 58% of allovarian neoplasms and more than 90% of malignant tumors of ovary.Debulking surgery and platinum-based combination chemotherapy (includingtaxanes) are current treatment modalities; however, the majority ofpatients with relapsed epithelial ovarian cancer eventually succumb tothe disease. There is a need for novel treatment modalities in ovariancancer, including targeted therapies such as immunotherapy withmonoclonal antibodies or cancer vaccine-based approaches.

NSCLC is any type of epithelial lung cancer other than small cell lungcarcinoma (SCLC). NSCLC accounts for about 85% of all lung cancers. As aclass, NSCLCs are relatively insensitive to chemotherapy, compared tosmall cell carcinoma. When possible, they are primarily treated bysurgical resection with curative intent, although chemotherapy isincreasingly being used both pre-operatively (neoadjuvant chemotherapy)and post-operatively (adjuvant chemotherapy). In the metastatic orinoperative setting, chemotherapy and/or immunotherapy is used, althoughthe disease at this stage is largely incurable and survival timesremains short. There is a need for novel treatment modalities in NSCLC,including targeted therapies such as immunotherapy with monoclonalantibodies or cancer vaccine-based approaches.

Accordingly, there exists a need for therapies that target thebiological activities of NaPi2b.

SUMMARY

The present disclosure provides a NaPi2b-targeted antibody-drugconjugate (such as a NaPi2b-targeted antibody-polymer-drug conjugate)that is biodegradable, biocompatible and exhibits high drug load as wellas specific binding to the extracellular region of SLC34A2. TheNaPi2b-targeted antibody-drug conjugates (e.g., NaPi2b-targetedantibody-polymer-drug conjugates) provided herein include an antibodythat specifically recognizes NaPi2b, also known as sodium-dependentphosphate transport protein 2B. The antibodies used in theNaPi2b-targeted antibody-drug conjugates disclosed herein can or mayalso include those that are capable of and useful for modulating,blocking, inhibiting, reducing, antagonizing, neutralizing or otherwiseinterfering with at least one biological activity of NaPi2b. Antibodiesdisclosed herein also include antibodies that bind soluble NaPi2b.

In some embodiments, the NaPi2b-targeted antibody disclosed herein canbe connected with an agent to form a conjugate. In some embodiments, theagent is a therapeutic agent. In some embodiments, the agent is anantineoplastic agent. In some embodiments, the agent is a toxin orfragment thereof. In some embodiments, the agent is (a) an auristatincompound; (b) a calicheamicin compound; (c) a duocarmycin compound; (d)SN38, (e) a pyrrolobenzodiazepine; (f) a vinca compound; (g) a tubulysincompound; (h) a non-natural camptothecin compound; (i) a maytansinoidcompound; (j) a DNA binding drug; (k) a kinase inhibitor; (1) a MEKinhibitor; (m) a KSP inhibitor; (n) a topoisomerase inhibitor; (o) aDNA-alkylating drug; (p) a RNA polymerase inhibitor; or analoguesthereof. In some embodiments, the agent is conjugated to theNaPi2b-targeted antibody via a linker. In some embodiments, the linkeris a cleavable linker. In some embodiments, the linker is anon-cleavable linker. In some embodiments, the agent is any of thetoxins described herein.

In one aspect, the NaPi2b-targeted antibody conjugate described hereinincludes an isolated NaPi2b-targeted antibody, connected directly orindirectly to one or more therapeutic or diagnostic agents (“D”). Insome embodiments, the NaPi2b-targeted antibody conjugate also includesone or more polymeric scaffolds connected to the antibody, wherein eachof the one or more D is independently connected to the antibody via theone or more polymeric scaffolds.

In some embodiments, each of the one or more polymeric scaffolds thatare connected to the isolated NaPi2b-targeted antibody, independently,comprises poly(l-hydroxymethylethylene hydroxymethyl-formal) (PHF),e.g., PHF having a molecular weight ranging from about 2 kDa to about 40kDa.

In some embodiments, each of the one or more polymeric scaffoldsindependently is of Formula (Ic):

wherein:

L^(D1) is a carbonyl-containing moiety;

each occurrence of

is independently a first linker that contains a biodegradable bond sothat when the bond is broken, D is released in an active form for itsintended therapeutic effect; and the

between L^(D1) and D denotes direct or indirect attachment of D toL^(D1);

each occurrence of

is independently a second linker not yet connected to the isolatedantibody, in which L^(P2) is a moiety containing a functional group thatis yet to form a covalent bond with a functional group of the isolatedantibody and the

between L^(D1) and L^(P2) denotes direct or indirect attachment ofL^(P2) to L^(D1), and each occurrence of the second linker is distinctfrom each occurrence of the first linker;

each occurrence of

is independently a third linker that connects each D-carrying polymericscaffold to the isolated antibody in which the terminal

attached to L^(P2) denotes direct or indirect attachment of L^(P2) tothe isolated antibody upon formation of a covalent bond between afunctional group of L^(P2) and a functional group of the isolatedantibody; and each occurrence of the third linker is distinct from eachoccurrence of the first linker;

m is an integer from 1 to about 300,

m₁ is an integer from 1 to about 140,

m₂ is an integer from 1 to about 40,

m₃ is an integer from 0 to about 18,

m₄ is an integer from 1 to about 10;

the sum of m, m₁, m₂, m₃, and m₄ ranges from about 15 to about 300; andthe total number of L^(P2) connected to the isolated antibody is 10 orless.

The conjugate described herein can include one or more of the followingfeatures:

For example, in Formula (Ic), the isolated NaPi2b-targeted antibody hasa molecular weight of 40 kDa or greater (e.g., 60 kDa or greater, 80 kDaor greater, 100 kDa or greater, 120 kDa or greater, 140 kDa or greater,160 kDa or greater, 180 kDa or greater, or 200 kDa or greater, or about40-200 kDa, 40-180 kDa, 40-140 kDa, 60-200 kDa, 60-180 kDa, 60-140 kDa,80-200 kDa, 80-180 kDa, 80-140 kDa, 100-200 kDa, 100-180 kDa, 100-140kDa, or 140-150 kDa). In some embodiments, the isolated NaPi2b-targetedantibody or any antibody of the disclosure, including, by way ofnon-limiting example, the XMT 1535 antibody and the 10H1.11.4B antibodydescribed herein.

For example, in Formula (Ic), the isolated NaPi2b-targeted antibodyspecifically binds to an epitope of human NaPi2b. In some embodiments,the isolated antibody specifically binds to an epitope on theextracellular domain of human NaPi2b.

For example, in Formula (Ic), the isolated NaPi2b-targeted antibody isthe XMT 1535 antibody and/or the 10H1.11.4B antibody described herein,as well as biosimilars thereof. Alternatively, the monoclonal antibodyis an antibody that binds to the same epitope and/or cross blocks anantibody of the disclosure or biosimilars thereof. These antibodies arerespectively referred to herein as “NaPi2b antibodies” or“NaPi2b-targeted antibodies”. NaPi2b antibodies include fully humanmonoclonal antibodies, as well as humanized monoclonal antibodies andchimeric antibodies. These antibodies show specificity for human NaPi2b,and they can or may modulate, block, inhibit, reduce, antagonize,neutralize or otherwise interfere with at least one NaPi2b activity.

For example, in Formula (Ic), the isolated NaPi2b-targeted antibody is amonoclonal antibody.

For example, in Formula (Ic), the isolated NaPi2b-targeted antibody is arabbit, mouse, chimeric, humanized or fully human monoclonal antibody.

For example, in Formula (Ic), the isolated NaPi2b-targeted antibody isan IgG isotype. In some embodiments, the isolated NaPi2b-targetedantibody is an IgG1 isotype.

For example, in Formula (Ic), the isolated NaPi2b-targeted antibodyincludes a variable heavy chain complementarity determining region 1(CDRH1) comprising an amino acid sequence at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequenceGYTFTGYNIH (SEQ ID NO: 5); a variable heavy chain complementaritydetermining region 2 (CDRH2) comprising an amino acid sequence at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to theamino acid sequence AIYPGNGDTSYKQKFRG (SEQ ID NO: 6); and a variableheavy chain complementarity determining region 3 (CDRH3) comprising anamino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%,99% or more identical to the amino acid sequence GETARATFAY (SEQ ID NO:7). For example, in Formula (Ic), the isolated NaPi2b-targeted antibodyincludes a variable light chain complementarity determining region 1(CDRL1) comprising an amino acid sequence at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequenceSASQDIGNFLN (SEQ ID NO: 8); a variable light chain complementaritydetermining region 2 (CDRL2) comprising an amino acid sequence at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to theamino acid sequence YTSSLYS (SEQ ID NO: 9); and a variable light chaincomplementarity determining region 3 (CDRL3) comprising an amino acidsequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% ormore identical to the amino acid sequence QQYSKLPLT (SEQ ID NO: 10).

For example, in Formula (Ic), the isolated NaPi2b-targeted antibodyincludes a CDRH1 comprising an amino acid sequence at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the aminoacid sequence GYTFTGYNIH (SEQ ID NO: 5); a CDRH2 comprising an aminoacid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99%or more identical to the amino acid sequence AIYPGNGDTSYKQKFRG (SEQ IDNO: 6); a CDRH3 comprising an amino acid sequence at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the aminoacid sequence GETARATFAY (SEQ ID NO: 7); a CDRL1 comprising an aminoacid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99%or more identical to the amino acid sequence SASQDIGNFLN (SEQ ID NO: 8);a CDRL2 comprising an amino acid sequence at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequenceYTSSLYS (SEQ ID NO: 9); and a CDRL3 comprising an amino acid sequence atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identicalto the amino acid sequence QQYSKLPLT (SEQ ID NO: 10).

For example, in Formula (Ic), the isolated NaPi2b-targeted antibodyincludes a variable heavy chain complementarity determining region 1(CDRH1) comprising the amino acid sequence GYTFTGYNIH (SEQ ID NO: 5); avariable heavy chain complementarity determining region 2 (CDRH2)comprising the amino acid sequence AIYPGNGDTSYKQKFRG (SEQ ID NO: 6); anda variable heavy chain complementarity determining region 3 (CDRH3)comprising the amino acid sequence GETARATFAY (SEQ ID NO: 7). Forexample, in Formula (Ic), the isolated NaPi2b-targeted antibody includesa variable light chain complementarity determining region 1 (CDRL1)comprising the amino acid sequence SASQDIGNFLN (SEQ ID NO: 8); avariable light chain complementarity determining region 2 (CDRL2)comprising the amino acid sequence YTSSLYS (SEQ ID NO: 9); and avariable light chain complementarity determining region 3 (CDRL3)comprising the amino acid sequence QQYSKLPLT (SEQ ID NO: 10).

For example, in Formula (Ic), the isolated NaPi2b-targeted antibodyincludes a CDRH1 comprising the amino acid sequence GYTFTGYNIH (SEQ IDNO: 5); a CDRH2 comprising the amino acid sequence AIYPGNGDTSYKQKFRG(SEQ ID NO: 6); a variable CDRH3 comprising the amino acid sequenceGETARATFAY (SEQ ID NO: 7); a CDRL1 comprising the amino acid sequenceSASQDIGNFLN (SEQ ID NO: 8); a CDRL2 comprising the amino acid sequenceYTSSLYS (SEQ ID NO: 9); and a CDRL3 comprising the amino acid sequenceQQYSKLPLT (SEQ ID NO: 10).

For example, in Formula (Ic), the isolated NaPi2b-targeted antibodyincludes a variable heavy chain (VH) region comprising an amino acidsequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% ormore identical to the amino acid sequence of SEQ ID NO: 3. For example,in Formula (Ic), the isolated NaPi2b-targeted antibody includes avariable light chain (VL) region comprising an amino acid sequence atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identicalto the amino acid sequence of SEQ ID NO: 4.

For example, in Formula (Ic), the isolated NaPi2b-targeted antibodyincludes a VH region comprising an amino acid sequence at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to theamino acid sequence of SEQ ID NO: 3, and a VL region comprising an aminoacid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99%or more identical to the amino acid sequence of SEQ ID NO: 4.

For example, in Formula (Ic), the isolated NaPi2b-targeted antibodyincludes a variable heavy chain (VH) region comprising the amino acidsequence of SEQ ID NO: 3. In some embodiments, the antibodies used inthe antibody-drug conjugates disclosed herein thereof used in theantibody-drug conjugates disclosed herein include a variable light chain(VL) region comprising the amino acid sequence of SEQ ID NO: 4.

For example, in Formula (Ic), the isolated NaPi2b-targeted antibodyincludes a VH region comprising the amino acid sequence of SEQ ID NO: 3,and a VL region comprising the amino acid sequence of SEQ ID NO: 4.

For example, in Formula (Ic), the isolated NaPi2b-targeted antibodyincludes a heavy chain comprising an amino acid sequence at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to theamino acid sequence of SEQ ID NO: 1. For example, in Formula (Ic), theisolated NaPi2b-targeted antibody includes a light chain comprising anamino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%,99% or more identical to the amino acid sequence of SEQ ID NO: 2.

For example, in Formula (Ic), the isolated NaPi2b-targeted antibodyincludes a heavy chain comprising an amino acid sequence at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to theamino acid sequence of SEQ ID NO: 1, and a light chain comprising anamino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%,99% or more identical to the amino acid sequence of SEQ ID NO: 2.

For example, in Formula (Ic), the isolated NaPi2b-targeted antibodyincludes a heavy chain comprising the amino acid sequence of SEQ IDNO: 1. For example, in Formula (Ic), the isolated NaPi2b-targetedantibody includes a light chain comprising the amino acid sequence ofSEQ ID NO: 2.

For example, in Formula (Ic), the isolated NaPi2b-targeted antibodyincludes a heavy chain comprising the amino acid sequence of SEQ ID NO:1, and a light chain comprising the amino acid sequence of SEQ ID NO: 2.

For example, in Formula (Ic), the isolated NaPi2b-targeted antibodyincludes a variable heavy chain complementarity determining region 1(CDRH1) comprising an amino acid sequence at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequenceGFSFSDFAMS (SEQ ID NO: 18); a variable heavy chain complementaritydetermining region 2 (CDRH2) comprising an amino acid sequence at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to theamino acid sequence ATIGRVAFHTYYPDSMKG (SEQ ID NO: 19); and a variableheavy chain complementarity determining region 3 (CDRH3) comprising anamino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%,99% or more identical to the amino acid sequence ARHRGFDVGHFDF (SEQ IDNO: 20). For example, in Formula (Ic), the isolated NaPi2b-targetedantibody includes a variable light chain complementarity determiningregion 1 (CDRL1) comprising an amino acid sequence at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the aminoacid sequence RSSETLVHSSGNTYLE (SEQ ID NO: 21); a variable light chaincomplementarity determining region 2 (CDRL2) comprising an amino acidsequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% ormore identical to the amino acid sequence RVSNRFS (SEQ ID NO: 22); and avariable light chain complementarity determining region 3 (CDRL3)comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97% 98%, 99% or more identical to the amino acid sequence FQGSFNPLT(SEQ ID NO: 23).

For example, in Formula (Ic), the isolated NaPi2b-targeted antibodyincludes a CDRH1 comprising an amino acid sequence at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the aminoacid sequence GFSFSDFAMS (SEQ ID NO: 18); a CDRH2 comprising an aminoacid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99%or more identical to the amino acid sequence ATIGRVAFHTYYPDSMKG (SEQ IDNO: 19); a CDRH3 comprising an amino acid sequence at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the aminoacid sequence ARHRGFDVGHFDF (SEQ ID NO: 20); a CDRL1 comprising an aminoacid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99%or more identical to the amino acid sequence RSSETLVHSSGNTYLE (SEQ IDNO: 21); a CDRL2 comprising an amino acid sequence at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the aminoacid sequence RVSNRFS (SEQ ID NO: 22); and a CDRL3 comprising an aminoacid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99%or more identical to the amino acid sequence FQGSFNPLT (SEQ ID NO: 23).

For example, in Formula (Ic), the isolated NaPi2b-targeted antibodyincludes a variable heavy chain complementarity determining region 1(CDRH1) comprising the amino acid sequence GFSFSDFAMS (SEQ ID NO: 18); avariable heavy chain complementarity determining region 2 (CDRH2)comprising the amino acid sequence ATIGRVAFHTYYPDSMKG (SEQ ID NO: 19);and a variable heavy chain complementarity determining region 3 (CDRH3)comprising the amino acid sequence ARHRGFDVGHFDF (SEQ ID NO: 20). Forexample, in Formula (Ic), the isolated NaPi2b-targeted antibody includesa variable light chain complementarity determining region 1 (CDRL1)comprising the amino acid sequence RSSETLVHSSGNTYLE (SEQ ID NO: 21); avariable light chain complementarity determining region 2 (CDRL2)comprising the amino acid sequence RVSNRFS (SEQ ID NO: 22); and avariable light chain complementarity determining region 3 (CDRL3)comprising the amino acid sequence FQGSFNPLT (SEQ ID NO: 23).

For example, in Formula (Ic), the isolated NaPi2b-targeted antibodyincludes a CDRH1 comprising the amino acid sequence GFSFSDFAMS (SEQ IDNO: 18); a CDRH2 comprising the amino acid sequence ATIGRVAFHTYYPDSMKG(SEQ ID NO: 19); a CDRH3 comprising the amino acid sequenceARHRGFDVGHFDF (SEQ ID NO: 20); a CDRL1 comprising the amino acidsequence RSSETLVHSSGNTYLE (SEQ ID NO: 21); a CDRL2 comprising the aminoacid sequence RVSNRFS (SEQ ID NO: 22); and a CDRL3 comprising the aminoacid sequence FQGSFNPLT (SEQ ID NO: 23).

For example, in Formula (Ic), the isolated NaPi2b-targeted antibodyincludes a variable heavy chain (VH) region comprising an amino acidsequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% ormore identical to the amino acid sequence of SEQ ID NO: 16. For example,in Formula (Ic), the isolated NaPi2b-targeted antibody includes avariable light chain (VL) region comprising an amino acid sequence atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identicalto the amino acid sequence of SEQ ID NO: 17.

For example, in Formula (Ic), the isolated NaPi2b-targeted antibodyincludes a VH region comprising an amino acid sequence at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to theamino acid sequence of SEQ ID NO: 16, and a VL region comprising anamino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%,99% or more identical to the amino acid sequence of SEQ ID NO: 17.

For example, in Formula (Ic), the isolated NaPi2b-targeted antibodyincludes a variable heavy chain (VH) region comprising the amino acidsequence of SEQ ID NO: 16. For example, in Formula (Ic), the isolatedNaPi2b-targeted antibody includes a variable light chain (VL) regioncomprising the amino acid sequence of SEQ ID NO: 17.

For example, in Formula (Ic), the isolated NaPi2b-targeted antibodyincludes a VH region comprising the amino acid sequence of SEQ ID NO:16, and a VL region comprising the amino acid sequence of SEQ ID NO: 17.

For example, in Formula (Ic), the isolated NaPi2b-targeted antibodyincludes a heavy chain comprising an amino acid sequence at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to theamino acid sequence of SEQ ID NO: 14. For example, in Formula (Ic), theisolated NaPi2b-targeted antibody includes a light chain comprising anamino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%,99% or more identical to the amino acid sequence of SEQ ID NO: 15.

For example, in Formula (Ic), the isolated NaPi2b-targeted antibodyincludes a heavy chain comprising an amino acid sequence at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to theamino acid sequence of SEQ ID NO: 14, and a light chain comprising anamino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%,99% or more identical to the amino acid sequence of SEQ ID NO: 15.

For example, in Formula (Ic), the isolated NaPi2b-targeted antibodyincludes a heavy chain comprising the amino acid sequence of SEQ ID NO:14. For example, in Formula (Ic), the isolated NaPi2b-targeted antibodyincludes a light chain comprising the amino acid sequence of SEQ ID NO:15.

For example, in Formula (Ic), the isolated NaPi2b-targeted antibodyincludes a heavy chain comprising the amino acid sequence of SEQ ID NO:14, and a light chain comprising the amino acid sequence of SEQ ID NO:15.

For example, in Formula (Ic), the isolated NaPi2b-targeted antibody isan isolated antibody that competes for specific binding to human NaPi2bwith an isolated antibody that includes (i) a variable heavy chaincomplementarity determining region 1 (CDRH1) comprising the amino acidsequence GYTFTGYNIH (SEQ ID NO: 5); a variable heavy chaincomplementarity determining region 2 (CDRH2) comprising the amino acidsequence AIYPGNGDTSYKQKFRG (SEQ ID NO: 6); a variable heavy chaincomplementarity determining region 3 (CDRH3) comprising the amino acidsequence GETARATFAY (SEQ ID NO: 7); a variable light chaincomplementarity determining region 1 (CDRL1) comprising the amino acidsequence SASQDIGNFLN (SEQ ID NO: 8); a variable light chaincomplementarity determining region 2 (CDRL2) comprising the amino acidsequence YTSSLYS (SEQ ID NO: 9); and a variable light chaincomplementarity determining region 3 (CDRL3) comprising the amino acidsequence QQYSKLPLT (SEQ ID NO: 10) or (ii) a CDRH1 comprising the aminoacid sequence GFSFSDFAMS (SEQ ID NO: 18); a CDRH2 comprising the aminoacid sequence ATIGRVAFHTYYPDSMKG (SEQ ID NO: 19); a CDRH3 comprising theamino acid sequence ARHRGFDVGHFDF (SEQ ID NO: 20); a CDRL1 comprisingthe amino acid sequence RSSETLVHSSGNTYLE (SEQ ID NO: 21); a CDRL2comprising the amino acid sequence RVSNRFS (SEQ ID NO: 22); and a CDRL3comprising the amino acid sequence FQGSFNPLT (SEQ ID NO: 23).

For example, in Formula (Ic), m₁ is an integer from 1 to about 120(e.g., about 1-90) and/or m₃ is an integer from 1 to about 10 (e.g.,about 1-8).

For example, when the PHF in Formula (Ic) has a molecular weight rangingfrom about 6 kDa to about 20 kDa (i.e., the sum of m, m₁, m₂, m₃, and m₄ranging from about 45 to about 150), m₂ is an integer from 2 to about20, m₃ is an integer from 0 to about 9, m₄ is an integer from 1 to about10, and/or m₁ is an integer from 1 to about 75 (e.g., m₁ being about4-45).

For example, when the PHF in Formula (Ic) has a molecular weight rangingfrom about 8 kDa to about 15 kDa (i.e., the sum of m, m₁, m₂, m₃, and m₄ranging from about 60 to about 110), m₂ is an integer from 2 to about15, m₃ is an integer from 0 to about 7, m₄ is an integer from 1 to about10, and/or m₁ is an integer from 1 to about 55 (e.g., m₁ being about4-30).

For example, when the PHF in Formula (Ic) has a molecular weight rangingfrom about 2 kDa to about 20 kDa (i.e., the sum of m, m₁, m₂, m₃, and m₄ranging from about 15 to about 150), m₂ is an integer from 1 to about20, m₃ is an integer from 0 to about 10 (e.g., m₃ ranging from 0 toabout 9), 1114 is an integer from 1 to about 8, and/or m₁ is an integerfrom 1 to about 70, and the total number of L¹² connected to theisolated antibody ranges from about 2 to about 8 (e.g., about 2, 3, 4,5, 6, 7, or 8).

For example, when the PHF in Formula (Ic) has a molecular weight rangingfrom about 3 kDa to about 15 kDa (i.e., the sum of m, m₁, m₂, m₃, and m₄ranging from about 20 to about 110), m₂ is an integer from 2 to about15, m₃ is an integer from 0 to about 8 (e.g., m₃ ranging from 0 to about7), m₄ is an integer from 1 to about 8, and/or m₁ is an integer from 2to about 50, and the total number of L¹² connected to the isolatedantibody ranges from about 2 to about 8 (e.g., about 2, 3, 4, 5, 6, 7,or 8).

For example, when the PHF in Formula (Ic) has a molecular weight rangingfrom about 5 kDa to about 10 kDa, (i.e., the sum of m, m₁, m₂, m₃ and m₄ranges from about 40 to about 75), m₂ is an integer from about 2 toabout 10 (e.g., m₂ being about 3-10), m₃ is an integer from 0 to about 5(e.g., m₃ ranging from 0 to about 4), m₄ is an integer from 1 to about 8(e.g., m₄ ranging from 1 to about 5), and/or m₁ is an integer from about2 to about 35 (e.g., m₁ being about 5-35), and the total number of L¹²connected to the isolated antibody ranges from about 2 to about 8 (e.g.,about 2, 3, 4, 5, 6, 7, or 8).

For example, each occurrence of D independently is a therapeutic agent,e.g., having a molecular weight of ≤5 kDa.

For example, each occurrence of D independently is a diagnostic agent ora label.

For example, some occurrences of D independently are therapeutic agents(e.g., having a molecular weight of ≤5 kDa) and the other occurrences ofD are diagnostic agents or labels.

For example, each occurrence of D independently is an anti-cancer drug,for example, selected from vinca alkaloids, auristatins, tubulysins,duocarmycins, non-natural camptothecin compounds, maytansinoids,calicheamicin compounds, topoisomerase inhibitors,pyrrolobenzodiazepines, DNA binding drugs, DNA-alkylating drugs, RNApolymerase inhibitors, kinase inhibitors, MEK inhibitors, KSPinhibitors, and analogs thereof.

For example, each occurrence of the auristatin compound is auristatin,dolastatin (e.g., dolastatin 10 or dolastatin 15), monomethylauristatinE (MMAE), monomethylauristatin F (MMAF), auristatin F hydroxypropylamide (AF HPA), monomethylauristatin F hydroxypropyl amide (AF HPA), orauristatin F phenylenediamine (AFP).

For example, each occurrence of the duocarmycin or analog thereof isduocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1,duocarmycin C2, duocarmycin D, duocarmycin SA, CC-1065, adozelesin,bizelesin, or carzelesin.

For example, each occurrence of the camptothecin compound iscamptothecin, CPT-11 (irinotecan), SN-38, or topotecan.

For example, each occurrence of the pyrrolobenzodiazepine compound is apyrrolobenzodiazepine monomer, a symmetrical pyrrolobenzodiazepine dimeror an unsymmetrical pyrrolobenzodiazepine dimer.

For example, each

when not connected to the isolated antibody, independently comprises aterminal group W^(P), in which each W^(P) independently is:

in which R^(1K) is a leaving group (e.g., halide or RC(O)O— in which Ris hydrogen, an aliphatic, heteroaliphatic, carbocyclic, orheterocycloalkyl moiety), R^(1A) is a sulfur protecting group, and ringA is cycloalkyl or heterocycloalkyl, and R′ is hydrogen, an aliphatic,heteroaliphatic, carbocyclic, or heterocycloalkyl moiety.

For example, each R^(1A) independently is

in which r is 1 or 2 and each of R^(s1), R^(s2), and R^(s3) is hydrogen,an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety.

For example, the functional group of L^(P2) that is yet to form acovalent bond with a functional group of the isolated antibody (such asa functional group or a reactive moiety on an amino acid residue of theantibody, for example, a functional group on a cysteine residue or alysine residue of the antibody), is selected from —SR^(p), —S—S-LG,

and halo, in which LG is a leaving group, R^(p) is H or a sulfurprotecting group, and one of X_(a) and X_(b) is H and the other is awater-soluble maleimido blocking moiety, or X_(a) and X_(b), togetherwith the carbon atoms to which they are attached for a carbon-carbondouble bond. For example, the functional group of L^(P2) that is yet toform a covalent bond is a functional group that is not reacted with afunctional group of the isolated antibody, e.g.,

as the functional group of L^(P2), in which one of X_(a) and X_(b) is Hand the other is a water-soluble maleimido blocking moiety, or X_(a) andX_(b).

For example, L^(D1) comprises —X—(CH₂)_(v)—C(═O)— with X directlyconnected to the carbonyl group of

in which X is CH₂, O, or NH, and v is an integer from 1 to 6.

For example, each occurrence of

is independently—C(═O)—X—(CH₂)_(v)—C(═O)—NH—(CH₂)_(u)—NH—C(═O)—(CH₂)_(w)—(OCH2)_(x)—NHC(═O)—(CH₂)_(y)-M,in which X is CH₂, O, or NH, each of v, u, w, x and y independently isan integer from 1 to 6, and M is

wherein one of X_(a) and X_(b) is H and the other is a water-solublemaleimido blocking moiety, or X_(a) and X_(b), together with the carbonatoms to which they are attached for a carbon-carbon double bond.

For example, each of v, u, w, x and y is 2.

For example, the ratio between D and the isolated NaPi2b-targetedantibody is about 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1,16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6;1, 5:1, 4:1,3:1, 2:1 or 1:1.

For example, the ratio between D and the isolated NaPi2b-targetedantibody is about 20:1, 15:1, 10:1, 5:1, 2:1 or 1:1.

For example, the ratio between D and the isolated NaPi2b-targetedantibody is about 16:1, 15:1, 14:1, 13:1, 12:1, 11:1 or 10:1.

For example, the ratio between D and the isolated NaPi2b-targetedantibody is about 15:1, 14:1, 13:1, 12:1 or 11:1.

For example, the ratio between D and the isolated NaPi2b-targetedantibody is about 15:1, 14:1, 13:1 or 12:1.

For example, the ratio between the D and the isolated NaPi2b-targetedantibody is about 6:1, 5:1, 4:1, 3:1, 2:1 or 1:1.

For example, each of the one or more D-carrying polymeric scaffoldsindependently is of Formula (Id):

wherein:

m_(3a) is an integer from 0 to about 17,

m_(3b) is an integer from 1 to about 8, and

the terminal

denotes the direct attachment of the one or more polymeric scaffolds tothe isolated NaPi2b-targeted antibody having a molecular weight of 40kDa or greater and (i) a variable heavy chain complementaritydetermining region 1 (CDRH1) comprising the amino acid sequenceGYTFTGYNIH (SEQ ID NO: 5); a variable heavy chain complementaritydetermining region 2 (CDRH2) comprising the amino acid sequenceAIYPGNGDTSYKQKFRG (SEQ ID NO: 6); a variable heavy chain complementaritydetermining region 3 (CDRH3) comprising the amino acid sequenceGETARATFAY (SEQ ID NO: 7); a variable light chain complementaritydetermining region 1 (CDRL1) comprising the amino acid sequenceSASQDIGNFLN (SEQ ID NO: 8); a variable light chain complementaritydetermining region 2 (CDRL2) comprising the amino acid sequence YTSSLYS(SEQ ID NO: 9); and a variable light chain complementarity determiningregion 3 (CDRL3) comprising the amino acid sequence QQYSKLPLT (SEQ IDNO: 10) or (ii) a CDRH1 comprising the amino acid sequence GFSFSDFAMS(SEQ ID NO: 18); a CDRH2 comprising the amino acid sequenceATIGRVAFHTYYPDSMKG (SEQ ID NO: 19); a CDRH3 comprising the amino acidsequence ARHRGFDVGHFDF (SEQ ID NO: 20); a CDRL1 comprising the aminoacid sequence RSSETLVHSSGNTYLE (SEQ ID NO: 21); a CDRL2 comprising theamino acid sequence RVSNRFS (SEQ ID NO: 22); and a CDRL3 comprising theamino acid sequence FQGSFNPLT (SEQ ID NO: 23).

In some embodiments, the isolated NaPi2b-targeted antibody has amolecular weight of 40 kDa or greater and includes (i) a CDRH1comprising the amino acid sequence GYTFTGYNIH (SEQ ID NO: 5); a CDRH2comprising the amino acid sequence AIYPGNGDTSYKQKFRG (SEQ ID NO: 6); aCDRH3 comprising the amino acid sequence GETARATFAY (SEQ ID NO: 7); aCDRL1 comprising the amino acid sequence SASQDIGNFLN (SEQ ID NO: 8); aCDRL2 comprising the amino acid sequence YTSSLYS (SEQ ID NO: 9); and aCDRL3 comprising the amino acid sequence QQYSKLPLT (SEQ ID NO: 10).

In some embodiments, the isolated NaPi2b-targeted antibody has amolecular weight of 40 kDa or greater and includes a CDRH1 comprisingthe amino acid sequence GFSFSDFAMS (SEQ ID NO: 18); a CDRH2 comprisingthe amino acid sequence ATIGRVAFHTYYPDSMKG (SEQ ID NO: 19); a CDRH3comprising the amino acid sequence ARHRGFDVGHFDF (SEQ ID NO: 20); aCDRL1 comprising the amino acid sequence RSSETLVHSSGNTYLE (SEQ ID NO:21); a CDRL2 comprising the amino acid sequence RVSNRFS (SEQ ID NO: 22);and a CDRL3 comprising the amino acid sequence FQGSFNPLT (SEQ ID NO:23).

The scaffold of Formula (Id) can include one or more of the followingfeatures:

The sum of m_(3a) and m_(3b) is between 1 and 18.

When the PHF in Formula (Id) has a molecular weight ranging from about 2kDa to about 40 kDa, the sum of m, m₁, m₂, m_(3a) and m_(3b) ranges fromabout 15 to about 300, m₁ is an integer from 1 to about 140, m₂ is aninteger from 1 to about 40, m_(3a) is an integer from 0 to about 17,m_(3b) is an integer from 1 to about 8, the sum of m_(3a) and m_(3b)ranges from 1 and about 18, and the ratio between the PHF and theisolated NaPi2b-targeted antibody is 10 or less.

When the PHF in Formula (Id) has a molecular weight ranging from about 2kDa to about 20 kDa, the sum of m, m₁, m₂, m_(3a) and m_(3b) ranges fromabout 15 to about 150, m₁ is an integer from 1 to about 70, m₂ is aninteger from 1 to about 20, m_(3a) is an integer from 0 to about 9,m_(3b) is an integer from 1 to about 8, the sum of m_(3a) and m_(3b)ranges from 1 and about 10, and the ratio between the PHF and theisolated NaPi2b-targeted antibody is an integer from 2 to about 8.

When the PHF in Formula (Id) has a molecular weight ranging from about 3kDa to about 15 kDa, the sum of m, m₁, m₂, m_(3a) and m_(3b) ranges fromabout 20 to about 110, m₁ is an integer from 2 to about 50, m₂ is aninteger from 2 to about 15, m_(3a) is an integer from 0 to about 7,m_(3b) is an integer from 1 to about 8, the sum of m_(3a) and m_(3b)ranges from 1 and about 8; and the ratio between the PHF and theisolated NaPi2b-targeted antibody is an integer from 2 to about 8 (e.g.,from about 2 to about 6 or from about 2 to about 4).

When the PHF in Formula (Id) has a molecular weight ranging from about 5kDa to about 10 kDa, the sum of m, m₁, m₂, m_(3a) and m_(3b) ranges fromabout 40 to about 75, m₁ is an integer from about 2 to about 35, m₂ isan integer from about 2 to about 10, m_(3a) is an integer from 0 toabout 4, m_(3b) is an integer from 1 to about 5, the sum of m_(3a) andm_(3b) ranges from 1 and about 5; and the ratio between the PHF and theisolated NaPi2b-targeted antibody is an integer from 2 to about 8 (e.g.,from about 2 to about 6 or from about 2 to about 4).

In certain embodiments, the ratio between auristatin F hydroxypropylamide (“AF HPA”) and the isolated NaPi2b-targeted antibody can be about30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1,18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1 or6:1.

In certain embodiments, the ratio between AF HPA and the isolatedNaPi2b-targeted antibody can be about 25:1, 24:1, 23:1, 22:1, 21:1,20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1,8:1, 7:1 or 6:1.

In other embodiments, the ratio between AF HPA and the isolatedNaPi2b-targeted antibody can be about 20:1, 19:1, 18:1, 17:1, 16:1,15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1 or 6:1.

In some embodiments, the ratio between AF HPA and isolatedNaPi2b-targeted antibody can be about 16:1, 15:1, 14:1, 13:1, 12:1, 11:1or 10:1.

In some embodiments, the ratio between AF HPA and isolatedNaPi2b-targeted antibody can be about 15:1, 14:1, 13:1, 12:1 or 11:1.

In some embodiments, the ratio between AF HPA and isolatedNaPi2b-targeted antibody can be about 15:1, 14:1, 13:1 or 12:1.

In certain embodiments, the ratio between PHF and isolatedNaPi2b-targeted antibody can be about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1,4:1, 3:1, 2:1 or 1:1.

In certain embodiments, the ratio between PHF and isolatedNaPi2b-targeted antibody can be about 8:1, 7:1, 6:1, 5:1, 4:1, 3:1 or2:1.

In other embodiments, the ratio between PHF and isolated NaPi2b-targetedantibody can be about 6:1, 5:1, 4:1, 3:1, 2:1 or 1:1.

In other embodiments, the ratio between PHF and isolated NaPi2b-targetedantibody can be about 6:1, 5:1, 4:1, 3:1 or 2:1.

In other embodiments, the ratio between PHF and isolated NaPi2b-targetedantibody can be about 6:1, 5:1, 4:1 or 3:1.

In some embodiments, the ratio between PHF and isolated NaPi2b-targetedantibody can be about 5:1, 4:1 or 3:1.

In some embodiments, the ratio between PHF and isolated NaPi2b-targetedantibody can be about 4:1, 3:1 or 2:1.

The water-soluble maleimido blocking moieties (e.g., X_(a) or X_(b)) aremoieties that can be covalently attached to one of the two olefin carbonatoms upon reaction of the maleimido group with a thiol-containingcompound of Formula (II):

R₉₀—(CH₂)_(d)—SH   (II)

wherein:

R₉₀ is NHR₉₁, OH, COOR₉₃, CH(NHR₉₁)COOR₉₃ or a substituted phenyl group;

R₉₃ is hydrogen or C₁₋₄ alkyl;

R₉₁ is hydrogen, CH₃ or CH₃CO and

d is an integer from 1 to 3.

In one embodiment, the water-soluble maleimido blocking compound ofFormula (II) can be cysteine, N-acetyl cysteine, cysteine methyl ester,N-methyl cysteine, 2-mercaptoethanol, 3-mercaptopropanoic acid,2-mercaptoacetic acid, mercaptomethanol (i.e., HOCH₂SH), benzyl thiol inwhich phenyl is substituted with one or more hydrophilic substituents,or 3-aminopropane-1-thiol. The one or more hydrophilic substituents onphenyl comprise OH, SH, methoxy, ethoxy, COOH, CHO, COC₁₋₄ alkyl, NH₂,F, cyano, SO₃H, POSH, and the like.

In another aspect, the water-soluble maleimido blocking group is—S—(CH₂)_(d)—R₉₀, in which,

R₉₀ is OH, COOH, or CH(NHR₉₁)COOR₉₃;

R₉₃ is hydrogen or CH₃;

R₉₁ is hydrogen or CH₃CO; and

d is 1 or 2.

In another embodiment, the water-soluble maleimido blocking group is—S—CH₂—CH(NH₂)COOH.

In certain embodiments, the conjugate described herein comprises one ormore D-carrying PHF, each of which independently is of Formula (If),wherein the PHF has a molecular weight ranging from about 2 kDa to about40 kDa:

wherein:

m is an integer from 1 to about 300,

m₁ is an integer from 1 to about 140,

m₂ is an integer from 1 to about 40,

m_(3a) is an integer from 0 to about 17,

m_(3b) is an integer from 1 to about 8;

the sum of m_(3a) and m_(3b) ranges from 1 and about 18;

the sum of m, m₁, m₂, m_(3a), and m_(3b) ranges from about 15 to about300;

the terminal

denotes the attachment of one or more PHF polymeric scaffolds to theisolated antibody that specifically binds to SLC34A2, wherein theisolated antibody that specifically binds to SLC34A2 is an isolatedantibody that comprises (i) a variable heavy chain complementaritydetermining region 1 (CDRH1) comprising the amino acid sequenceGYTFTGYNIH (SEQ ID NO: 5); a variable heavy chain complementaritydetermining region 2 (CDRH2) comprising the amino acid sequenceAIYPGNGDTSYKQKFRG (SEQ ID NO: 6); a variable heavy chain complementaritydetermining region 3 (CDRH3) comprising the amino acid sequenceGETARATFAY (SEQ ID NO: 7); a variable light chain complementaritydetermining region 1 (CDRL1) comprising the amino acid sequenceSASQDIGNFLN (SEQ ID NO: 8); a variable light chain complementaritydetermining region 2 (CDRL2) comprising the amino acid sequence YTSSLYS(SEQ ID NO: 9); and a variable light chain complementarity determiningregion 3 (CDRL3) comprising the amino acid sequence QQYSKLPLT (SEQ IDNO: 10) or (ii) a CDRH1 comprising the amino acid sequence GFSFSDFAMS(SEQ ID NO: 18); a CDRH2 comprising the amino acid sequenceATIGRVAFHTYYPDSMKG (SEQ ID NO: 19); a CDRH3 comprising the amino acidsequence ARHRGFDVGHFDF (SEQ ID NO: 20); a CDRL1 comprising the aminoacid sequence RSSETLVHSSGNTYLE (SEQ ID NO: 21); a CDRL2 comprising theamino acid sequence RVSNRFS (SEQ ID NO: 22); and a CDRL3 comprising theamino acid sequence FQGSFNPLT (SEQ ID NO: 23); and the ratio between thePHF and the antibody is 10 or less.

In some embodiments, the isolated NaPi2b-targeted antibody specificallybinds to SLC34A2 and includes (i) a CDRH1 comprising the amino acidsequence GYTFTGYNIH (SEQ ID NO: 5); a CDRH2 comprising the amino acidsequence AIYPGNGDTSYKQKFRG (SEQ ID NO: 6); a CDRH3 comprising the aminoacid sequence GETARATFAY (SEQ ID NO: 7); a CDRL1 comprising the aminoacid sequence SASQDIGNFLN (SEQ ID NO: 8); a CDRL2 comprising the aminoacid sequence YTSSLYS (SEQ ID NO: 9); and a CDRL3 comprising the aminoacid sequence QQYSKLPLT (SEQ ID NO: 10).

In some embodiments, the isolated NaPi2b-targeted antibody specificallybinds to SLC34A2 and includes a CDRH1 comprising the amino acid sequenceGFSFSDFAMS (SEQ ID NO: 18); a CDRH2 comprising the amino acid sequenceATIGRVAFHTYYPDSMKG (SEQ ID NO: 19); a CDRH3 comprising the amino acidsequence ARHRGFDVGHFDF (SEQ ID NO: 20); a CDRL1 comprising the aminoacid sequence RSSETLVHSSGNTYLE (SEQ ID NO: 21); a CDRL2 comprising theamino acid sequence RVSNRFS (SEQ ID NO: 22); and a CDRL3 comprising theamino acid sequence FQGSFNPLT (SEQ ID NO: 23).

The scaffold of Formula (If) can include one or more of the followingfeatures:

When the PHF in Formula (If) has a molecular weight ranging from about 2kDa to about 20 kDa, the sum of m, m₁, m₂, m_(3a) and m_(3b) ranges fromabout 15 to about 150, m₁ is an integer from 1 to about 70, m₂ is aninteger from 1 to about 20, m_(3a) is an integer from 0 to about 9,m_(3b) is an integer from 1 to about 8, the sum of m_(3a) and m_(3b)ranges from 1 and about 10, and the ratio between the PHF and theantibody is an integer from 2 to about 8.

When the PHF in Formula (If) has a molecular weight ranging from about 3kDa to about 15 kDa, the sum of m, m₁, m₂, m_(3a) and m_(3b) ranges fromabout 20 to about 110, m₁ is an integer from 2 to about 50, m₂ is aninteger from 2 to about 15, m_(3a) is an integer from 0 to about 7,m_(3b) is an integer from 1 to about 8, the sum of m_(3a) and m_(3b)ranges from 1 and about 8; and the ratio between the PHF and theantibody is an integer from 2 to about 8 (e.g., from about 2 to about 6or from about 2 to about 4).

When the PHF in Formula (If) has a molecular weight ranging from about 5kDa to about 10 kDa, the sum of m, m₁, m₂, m_(3a) and m_(3b) ranges fromabout 40 to about 75, m₁ is an integer from about 2 to about 35, m₂ isan integer from about 2 to about 10, m_(3a) is an integer from 0 toabout 4, m_(3b) is an integer from 1 to about 5, the sum of m_(3a) andm_(3b) ranges from 1 and about 5; and the ratio between the PHF and theantibody is an integer from 2 to about 8 (e.g., from about 2 to about 6or from about 2 to about 4).

In certain embodiments, the ratio between auristatin F hydroxypropylamide (“AF HPA”) and the antibody can be about 30:1, 29:1, 28:1, 27:1,26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1,14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1 or 6:1.

In certain embodiments, the ratio between AF HPA and the antibody can beabout 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1,14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1 or 6:1.

In other embodiments, the ratio between AF HPA and the antibody can beabout 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1,9:1, 8:1, 7:1 or 6:1.

In some embodiments, the ratio between AF HPA and the antibody can beabout 16:1, 15:1, 14:1, 13:1, 12:1, 11:1 or 10:1.

In some embodiments, the ratio between AF and the antibody can be about15:1, 14:1, 13:1, 12:1 or 11:1.

In some embodiments, the ratio between AF HPA and the antibody can beabout 15:1, 14:1, 13:1 or 12:1.

In certain embodiments, the ratio between PHF and the antibody can beabout 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1 or 1:1.

In certain embodiments, the ratio between PHF and the antibody can beabout 8:1, 7:1, 6:1, 5:1, 4:1, 3:1 or 2:1.

In other embodiments, the ratio between PHF and the antibody can beabout 6:1, 5:1, 4:1, 3:1, 2:1 or 1:1.

In other embodiments, the ratio between PHF and the antibody can beabout 6:1, 5:1, 4:1, 3:1 or 2:1.

In other embodiments, the ratio between PHF and the antibody can beabout 6:1, 5:1, 4:1 or 3:1.

In some embodiments, the ratio between PHF and the antibody can be about5:1, 4:1 or 3:1.

In some embodiments, the ratio between PHF and the antibody can be about4:1, 3:1 or 2:1.

Another aspect of the disclosure features a method of preparing aconjugate described herein. The method includes reacting the isolatedantibody with a D-carrying polymeric scaffold of Formula (Ia) such thatthe conjugate is formed:

wherein:

L^(D1) is a carbonyl-containing moiety;

each occurrence of

is independently a first linker that contains a biodegradable bond sothat when the bond is broken, D is released in an active form for itsintended therapeutic effect; and the

between L^(D1) and D denotes direct or indirect attachment of D toL^(D1);

each occurrence of

is independently a second linker not yet connected to the isolatedantibody, in which L^(P2) is a moiety containing a functional group thatis yet to form a covalent bond with a functional group of the isolatedantibody, and the

between L^(D1) and L^(P2) denotes direct or indirect attachment ofL^(P2) to L^(D1), and each occurrence of the second linker is distinctfrom each occurrence of the first linker;

m is an integer from 1 to about 300,

m₁ is an integer from 1 to about 140,

m₂ is an integer from 1 to about 40,

m₃ is an integer from 1 to about 18, and

the sum of m, m₁, m₂ and m₃ ranges from about 15 to about 300.

In the formulae for polymeric scaffolds disclosed herein, thedisconnection or gap between the polyacetal units indicates that theunits can be connected to each other in any order. In other words, theappending groups that contain, e.g., D, L^(P2), and the isolatedantibody, can be randomly distributed along the polymer backbone.

The present disclosure also provides methods of treating, preventing,delaying the progression of or otherwise ameliorating a symptom of oneor more pathologies associated with aberrant NaPi2b expression, functionand/or activation or alleviating a symptom associated with suchpathologies, by administering a conjugate disclosed herein to a subjectin which such treatment or prevention is desired. The subject to betreated is, e.g., human. The conjugate are administered in an amountsufficient to treat, prevent or alleviate a symptom associated with thepathology.

The present disclosure also provides methods of treating, preventing,delaying the progression of or otherwise ameliorating a symptom of oneor more pathologies associated with NaPi2b expression, function and/oractivation or alleviating a symptom associated with such pathologies, byadministering a conjugate disclosed herein to a subject in which suchtreatment or prevention is desired. The subject to be treated is, e.g.,human. The conjugate is administered in an amount sufficient to treat,prevent or alleviate a symptom associated with the pathology.

Pathologies treated and/or prevented using the conjugates disclosedherein including, for example, a cancer. In some embodiments, theconjugates disclosed herein are useful in treating, preventing, delayingthe progression of or otherwise ameliorating a NaPi2b expressing cancer.For example, the conjugates disclosed herein are useful in treating,preventing, delaying the progression of or otherwise ameliorating asymptom of a cancer selected from the group consisting of ovarian cancer(such as epithelial ovarian cancer), thyroid cancer, colorectal cancer,lung cancer, non-small cell lung cancer (NSCLC) such as non-squamousNSCLC, breast cancer, kidney cancer and salivary duct carcinoma.

In some embodiments, the conjugates disclosed herein are useful intreating, preventing, delaying the progression of or otherwiseameliorating a symptom of ovarian cancer. In some embodiments, theovarian cancer is epithelial ovarian cancer.

In some embodiments, the conjugates disclosed herein are useful intreating, preventing, delaying the progression of or otherwiseameliorating a symptom of NSCLC. In some embodiments, the NSCLC isnon-squamous NSCLC.

In some embodiments, the conjugates disclosed herein are useful intreating, preventing, delaying the progression of or otherwiseameliorating a symptom of ovarian cancer. In some embodiments, theovarian cancer is epithelial ovarian cancer.

The disclosure also provides kits and/or methods for identifying orotherwise refining, e.g., stratifying, a patient population suitable fortherapeutic administration of a NaPi2b-targeted antibody-drug conjugatesdisclosed herein by identifying the NaPi2b score of subject prior totreatment with a NaPi2b-targeted antibody-drug conjugate disclosedherein. In some embodiments, the subject is identified as having ascoring of 1+ or 2+ or 3+ for NaPi2b expression. In some embodiments,the test cell population is derived from fresh, unfrozen tissue from abiopsy sample. In some embodiments, the test cell population is derivedfrom a primary or metastatic site. In some embodiments, the test cellpopulation is derived from a frozen tissue from a biopsy or surgicalsample or ascetic fluid or pleural fluid. In some embodiments, the testcell population is derived from a fixed tissue (e.g., formalin fixation)from a biopsy or surgical sample.

The IHC test measures the amount of NaPi2b receptor protein on thesurface of cells in a cancer tissue sample, e.g., an ovarian cancertissue sample or a lung cancer sample, and assigns the detected level ofcell surface NaPi2b receptor NaPi2b score of 0, 1+, 2+ or 3+.

In some embodiments, the subject is refractory to chemotherapy,including standard, front-line chemotherapeutic agents.

In some embodiments, the subject has platinum-resistant ovarian cancer.

In some embodiments, the subject has platinum-sensitive ovarian cancer.

In some embodiments, the subject has platinum-refractory ovarian cancer.

In some embodiments, the subject has advanced ovarian cancer and has notreceived any prior therapy for treating cancer (e.g., ovarian cancer).In some embodiments, the subject has advanced ovarian cancer and has notreceived any prior chemotherapy for treating cancer (e.g., ovariancancer).

The NaPi2b-targeted antibody-drug conjugates used in any of theembodiments of the methods and uses provided herein can be administeredat any stage of the disease. For example, such a NaPi2b-targetedantibody-drug conjugate can be administered to a patient sufferingcancer of any stage, from early to metastatic.

In some embodiments, the NaPi2b-targeted antibody-drug conjugates of thedisclosure can be administered either alone or in combination with othercompositions in a therapy. For instance, a conjugate of the disclosuremay be co-administered with at least one additional therapeutic agentand/or adjuvant. In certain embodiments, the additional therapeuticagent is a small molecule inhibitor, another antibody-based therapy, apolypeptide or peptide-based therapy, a nucleic acid-based therapyand/or other biologic. The additional therapeutic agent can either bethe same as the “D” used to form the conjugate or different.

In certain embodiments, the additional therapeutic agent is a cytotoxicagent, a chemotherapeutic agent, a growth inhibitory agent, anangiogenesis inhibitor, a PARP (poly(ADP)-ribose polymerase) inhibitor,an alkylating agent, an anti-metabolite, an anti-microtubule agent, atopoisomerase inhibitor, a cytotoxic antibiotic, any other nucleic aciddamaging agent or an immune checkpoint inhibitor. In one embodiment, thetherapeutic agent used in the treatment of cancer, includes but is notlimited to, a platinum compound (e.g., cisplatin or carboplatin); ataxane (e.g., paclitaxel or docetaxel); a topoisomerase inhibitor (e.g.,irinotecan or topotecan); an anthracycline (e.g., doxorubicin(ADRIAMYCIN®) or liposomal doxorubicin (DOXIL®)); an anti-metabolite(e.g., gemcitabine, pemetrexed); cyclophosphamide; vinorelbine(NAVELBINE®); hexamethylmelamine; ifosfamide; etoposide; an angiogenesisinhibitor (e.g., Bevacizumab (Avastin®)), thalidomide, TNP-470, plateletfactor 4, interferon or endostatin); a PARP inhibitor (e.g., Olaparib(Lynparza™)); an immune checkpoint inhibitor, such as for example, amonoclonal antibody that targets either PD-1 or PD-L ((e.g.,pembrolizumab (Keytruda®)), atezolizumab (MPDL3280A) or nivolumab(Opdivo®)) or CTLA-4 (e.g., Ipilimumab (Yervoy®)), a kinase inhibitor(e.g., sorafenib or erlotinib), an ALK inhibitor (e.g. crizotinib(Xalkori), ceritinib (Zykadia), alectinib (Alecensa), dalantercept(ACE-041), brigatinib (AP26113), entrectinib (NMS-E628), PF-06463922TSR-011, CEP-37440 and X-396), a proteasome inhibitor (e.g., bortezomibor carfilzomib), an immune modulating agent (e.g., lenalidomide orIL-2), a radiation agent, and/or a biosimilar thereof and/orcombinations thereof. Other suitable agents include an agent consideredstandard of care by those skilled in the art and/or a chemotherapeuticagent well known to those skilled in the art.

In some embodiments, the immune checkpoint inhibitor suitable for thecombinations and methods of the disclosure is a monoclonal antibody, ahumanized antibody, a fully human antibody, a fusion protein or acombination thereof.

In some embodiments, the immune checkpoint inhibitors inhibits acheckpoint protein that comprises CTLA-4, PDL1, PDL2, PD1, BTLA, HVEM,TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR,a B-7 family ligand, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86,CD137, CD226, CD276, DR3, GITR, HAVCR2, HVEM, IDO1, IDO2, ICOS(inducible T cell costimulator), LAIR1, LIGHT, MARCO (macrophagereceptor with collagenous structure), OX-40, SLAM, TIGHT, VTCN1 or acombination thereof.

In some embodiments, the immune checkpoint inhibitor interacts with aligand of a checkpoint protein that comprises CTLA-4, PDL1, PDL2, PD1,BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1,CHK2, A2aR, a B-7 family ligand, CD2, CD27, CD28, CD30, CD40, CD70,CD80, CD86, CD137, CD226, CD276, DR3, GITR, HAVCR2, HVEM, IDO1, IDO2,ICOS (inducible T cell costimulator), LAIR1, LIGHT, MARCO (macrophagereceptor with collagenous structure), OX-40, SLAM, TIGHT, VTCN1 or acombination thereof.

In some embodiments, the immune checkpoint inhibitor inhibits acheckpoint protein that comprises CTLA-4, PDL1, PD1 or a combinationthereof.

In some embodiments, the immune checkpoint inhibitor comprisespembrolizumab (MK-3475), nivolumab (BMS-936558), pidilizumab (CT-011),AMP-224, MDX-1 105, durvalumab (MEDI4736), MPDL3280A, BMS-936559,IPH2101, TSR-042, TSR-022, ipilimumab, lirilumab, atezolizumab,avelumab, tremelimumab, or a combination thereof.

In some embodiments, the immune checkpoint inhibitor comprises nivolumab(BMS-936558), ipilimumab, pembrolizumab, atezolizumab, tremelimumab,durvalumab, avelumab, or a combination thereof.

In some embodiments, the NaPi2b-targeted antibody-drug conjugate andadditional agent(s) are formulated into a single therapeuticcomposition, and the NaPi2b-targeted antibody-drug conjugate andadditional agent are administered simultaneously. Alternatively, theNaPi2b-targeted antibody-drug conjugate and additional agent areseparate from each other, e.g., each is formulated into a separatetherapeutic composition, and the NaPi2b-targeted antibody-drug conjugateand the additional agent are administered simultaneously, or theNaPi2b-targeted antibody-drug conjugates and the additional agent areadministered at different times during a treatment regimen. For example,the NaPi2b-targeted antibody-drug conjugate is administered prior to theadministration of the additional agent, the NaPi2b-targetedantibody-drug conjugate is administered subsequent to the administrationof the additional agent, or the NaPi2b-targeted antibody-drug conjugateand the additional agent are administered in an alternating fashion. Asdescribed herein, the NaPi2b-targeted antibody-drug conjugate antibodyand additional agent are administered in single doses or in multipledoses.

In some embodiments, the NaPi2b-targeted antibody-drug conjugate and theimmune checkpoint inhibitor are formulated in the same formulation.

In some embodiments, the NaPi2b-targeted antibody-drug conjugate and theimmune checkpoint inhibitor are formulated in separate formulations.

In some embodiments, the combination comprising a NaPi2b-targetedantibody-drug conjugate and an immune checkpoint inhibitor disclosedherein are useful in treating, preventing, delaying the progression ofor otherwise ameliorating a symptom of ovarian cancer. In someembodiments, the ovarian cancer is epithelial ovarian cancer.

In some embodiments, the combination comprising a NaPi2b-targetedantibody-drug conjugate and an immune checkpoint inhibitor disclosedherein are useful in treating, preventing, delaying the progression ofor otherwise ameliorating a symptom of NSCLC. In some embodiments, theNSCLC is non-squamous NSCLC.

Also disclosed are kits comprising a NaPi2b-targeted antibody-drugconjugate and an immune checkpoint inhibitor. The kit components may bepackaged together or separated into two or more containers. In someembodiments, the containers may be vials that contain sterile,lyophilized formulations of a composition that are suitable forreconstitution. A kit may also contain one or more buffers suitable forreconstitution and/or dilution of other reagents. Other containers thatmay be used include, but are not limited to, a pouch, tray, box, tube,or the like. Kit components may be packaged and maintained sterilelywithin the containers. Another component that can be included isinstructions to a person using a kit for its use.

Pharmaceutical compositions according to the disclosure can include aNaPi2b-targeted antibody-drug conjugate disclosed herein and a suitablecarrier. These pharmaceutical compositions can be included in kits, suchas, for example, diagnostic kits.

One skilled in the art will appreciate that the antibodies disclosedherein have a variety of uses. For example, the proteins disclosedherein are used as therapeutic agents. The antibodies disclosed hereinare also used as reagents in diagnostic kits or as diagnostic tools, orthese antibodies can be used in competition assays to generatetherapeutic reagents.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. In the specification, thesingular forms also include the plural unless the context clearlydictates otherwise. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent invention, suitable methods and materials are described below.In the case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods andexamples are illustrative only and are not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the anti-tumor efficacy of XMT-1535; Example 3A,rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))); Example 4A((10H1.11.4B)-(MC-VC-PABA-MMAE)); Example 2C ((10H1.11.4B)-(EG2-MI-(10kDa PHF-BA-(AF-HPA-Ala))) and Example 1B, XMT 1535-(EG2-MI-(10 kDaPHF-BA-(AF-HPA-Ala))), as measured in a OVCAR3 mouse tumor xenograftmodel.

FIG. 2 illustrates the anti-tumor efficacy of Example 3B,rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))); Example 4B((10H1.11.4B)-(MC-VC-PABA-MMAE)); and Example 1C, XMT 1535-(EG2-MI-(10kDa PHF-BA-(AF-HPA-Ala))) as measured in an OVCAR3 mouse tumor xenograftmodel.

FIG. 3 illustrates the anti-tumor efficacy of Example 3B,rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))); Example 4B((10H1.11.4B)-(MC-VC-PABA-MMAE)); and Example 1C, XMT 1535-(EG2-MI-(10kDa PHF-BA-(AF-HPA-Ala))), as measured in a KRAS mutant patient derivednon-small cell lung cancer xenograft model.

FIG. 4 illustrates the anti-tumor efficacy of Example 3B,rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))); Example 4B((10H1.11.4B)-(MC-VC-PABA-MMAE)); and Example 1C, XMT 1535-(EG2-MI-(10kDa PHF-BA-(AF-HPA-Ala))), as measured in a patient derived non-smallcell lung cancer xenograft model.

FIG. 5 illustrates the anti-tumor efficacy of Example 1C, XMT1535-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), as measured in a patientderived non-small cell lung cancer xenograft model.

FIGS. 6A and 6B respectively show the plasma pharmacokinetics for thetotal antibody and the total drug after administration of Example 1C at1.25 mg/kg (1074 μg/m² auristatin payload equivalents), 2.5 mg/kg (2147μg/m² auristatin payload equivalents) or 5 mg/kg (or 4294 μg/m²auristatin payload equivalents) as a single IV infusion in cynomolgusmonkeys.

FIG. 7 illustrates the anti-tumor efficacy of Example 3B,rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))); Example 4B((10H1.11.4B)-(MC-VC-PABA-MMAE)); and Example 1C, XMT 1535-(EG2-MI-(10kDa PHF-BA-(AF-HPA-Ala))), as measured in a patient derived non-smallcell lung cancer xenograft model.

FIG. 8 illustrates the anti-tumor efficacy of Example 1C, XMT1535-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), as measured in a patientderived non-small cell lung cancer xenograft model.

FIG. 9 illustrates the anti-tumor efficacy of Example 1C, XMT1535-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), as measured in a patientderived non-small cell lung cancer xenograft model.

FIG. 10 illustrates the anti-tumor efficacy of Example 1C, XMT1535-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), as measured in a patientderived non-small cell lung cancer xenograft model.

DETAILED DESCRIPTION

The present disclosure provides a NaPi2b-targeted antibody-drugconjugate (such as NaPi2b antibody-polymer-drug conjugate) that isbiodegradable, biocompatible and exhibits high drug load as well asspecifically binding to the extracellular region of SLC34A2. TheNaPi2b-targeted antibody-drug conjugates (such as NaPi2bantibody-polymer-drug conjugates) provided herein include an antibodythat specifically recognizes NaPi2b, also known as sodium-dependentphosphate transport protein 2B. The NaPi2b antibodies used in theconjugates disclosed herein are capable of and useful in modulating,e.g., blocking, inhibiting, reducing, antagonizing, neutralizing orotherwise interfering with at least one biological activity of NaPi2b.Antibodies disclosed herein also include antibodies that bind solubleNaPi2b.

The NaPi2b antibody-drug conjugates provided herein include antibodiesthat bind to a NaPi2b epitope with an equilibrium dissociation constant(K_(d) or K_(D)) of ≤1 μM, e.g., ≤100 nM, preferably ≤10 nM, and morepreferably ≤1 nM. For example, the NaPi2b antibodies used in theantibody-drug conjugates disclosed herein exhibit a K_(d) in the rangeapproximately between ≤1 nM to about 1 pM.

The NaPi2b antibody-drug conjugates provided herein can includeantibodies that serve to modulate, block, inhibit, reduce, antagonize,neutralize or otherwise interfere with the functional activity ofNaPi2b. Functional activities of NaPi2b include for example,participating in the transcellular inorganic phosphate (Pi) absorption,thereby contributing to the maintenance of phosphate homeostasis in thebody. For example, the NaPi2b antibodies completely or partially inhibitNaPi2b functional activity by partially or completely modulating,blocking, inhibiting, reducing antagonizing, neutralizing, or otherwiseinterfering with transcellular inorganic phosphate absorption.Transcellular inorganic phosphate absorption activity is assessed usingany art-recognized method for detecting transcellular inorganicphosphate absorption activity, including, but not limited to detectinglevels of transcellular inorganic phosphate absorption in the presenceand absence of an anti-NaPi2b antibody disclosed herein.

The NaPi2b antibodies used in the antibody-drug conjugates disclosedherein can be those that are considered to completely modulate, block,inhibit, reduce, antagonize, neutralize or otherwise interfere withNaPi2b functional activity when the level of NaPi2b functional activityin the presence of the NaPi2b antibody is decreased by at least 95%,e.g., by 96%, 97%, 98%, 99% or 100% as compared to the level of NaPi2bfunctional activity in the absence of binding with a NaPi2b antibodydescribed herein. The NaPi2b antibodies are considered to partiallymodulate, block, inhibit, reduce, antagonize, neutralize or otherwiseinterfere with NaPi2b functional activity when the level of NaPi2bactivity in the presence of the NaPi2b antibody is decreased by lessthan 95%, e.g., 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85% or 90%as compared to the level of NaPi2b activity in the absence of bindingwith a NaPi2b antibody described herein.

Unless otherwise defined, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures utilized in connection with, and techniques of, cell andtissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. See e.g., Sambrook etal. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclaturesutilized in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor chemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

As used herein, the terms “NaPi2b” (also known as sodium-dependentphosphate transport protein 2B, SLC34A2, NaPiIIb, Npt2, Na(+)-dependentphosphate cotransporter 2B; sodium/phosphate cotransporter 2B; Na(+)/Picotransporter 2B; NaPi3b; solute carrier family 34 member 2), when usedherein, refers to human NaPi2b (e.g., GenBank Accession No. 095436.3)and includes any variants, isoforms and species homologs of NaPi2b whichare naturally expressed by cells, including tumor cells, or areexpressed on cells transfected with the NaPi2b gene. These terms aresynonymous and may be used interchangeably.

As used herein, the term “NaPi2b antibody” or “anti-NaPi2b antibody” isan antibody that binds specifically to the antigen NaPi2b.

When used herein in the context of two or more antibodies, the term“competes with” or “cross-competes with” indicates that the two or moreantibodies compete for binding to NaPi2b, e.g., compete for NaPi2bbinding in any art-recognized assay. An antibody “blocks” or“cross-blocks” one or more other antibodies from binding to NaPi2b ifthe antibody competes with the one or more other antibodies 25% or more,with 25%-74% representing “partial block” and 75%-100% representing“full block”, as determined using any art-recognized assay. For somepairs of antibodies, competition or blocking in any art-recognized assayis only observed when one antibody is coated on the plate and the otheris used to compete, and not vice versa. Unless otherwise defined ornegated by context, the terms “competes with”, “cross-competes with”,“blocks” or “cross-blocks” when used herein is also intended to coversuch pairs of antibodies.

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin (Ig) molecules,i.e., molecules that contain an antigen binding site that specificallybinds (immunoreacts with) an antigen. By “specifically bind” or“immunoreacts with” “or directed against” is meant that the antibodyreacts with one or more antigenic determinants of the desired antigenand does not react with other polypeptides or binds at much loweraffinity (K_(d)>10⁻⁶). Antibodies include, but are not limited to,polyclonal, monoclonal and chimeric antibodies.

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Ingeneral, antibody molecules obtained from humans relate to any of theclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses as well, such as IgG₁, IgG₂, and others. Furthermore, inhumans, the light chain may be a kappa chain or a lambda chain.

The term “monoclonal antibody” (mAb) or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one molecular species of antibody moleculeconsisting of a unique light chain gene product and a unique heavy chaingene product. In particular, the complementarity determining regions(CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. Monoclonal antibodies (mAbs) contain an antigen-bindingsite capable of immunoreacting with a particular epitope of the antigencharacterized by a unique binding affinity for it.

In general, antibody molecules obtained from humans relate to any of theclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses as well, such as IgG₁, IgG₂, and others. Furthermore, inhumans, the light chain may be a kappa chain or a lambda chain.

The term “antigen-binding site” or “binding portion” refers to the partof the immunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains, referred to as “hypervariable regions,” are interposed betweenmore conserved flanking stretches known as “framework regions,” or“FRs”. Thus, the term “FR” refers to amino acid sequences that arenaturally found between, and adjacent to, hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen-binding surface. The antigen-binding surface iscomplementary to the three-dimensional surface of a bound antigen, andthe three hypervariable regions of each of the heavy and light chainsare referred to as “complementarity-determining regions,” or “CDRs.” Theassignment of amino acids to each domain is in accordance with thedefinitions of Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987 and 1991)), orChothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature342:878-883 (1989).

As used herein, the term “epitope” includes any protein determinantcapable of specific binding to an immunoglobulin or fragment thereof, ora T-cell receptor. The term “epitope” includes any protein determinantcapable of specific binding to an immunoglobulin or T-cell receptor.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics. An antibody is said tospecifically bind an antigen when the dissociation constant is ≤1 μM;e.g., ≤100 nM, preferably ≤10 nM and more preferably ≤1 nM.

As used herein, the terms “immunological binding,” and “immunologicalbinding properties” refer to the non-covalent interactions of the typewhich occur between an immunoglobulin molecule and an antigen for whichthe immunoglobulin is specific. The strength, or affinity ofimmunological binding interactions can be expressed in terms of thedissociation constant (K_(d)) of the interaction, wherein a smallerK_(d) represents a greater affinity. Immunological binding properties ofselected polypeptides can be quantified using methods well known in theart. One such method entails measuring the rates of antigen-bindingsite/antigen complex formation and dissociation, wherein those ratesdepend on the concentrations of the complex partners, the affinity ofthe interaction, and geometric parameters that equally influence therate in both directions. Thus, both the “on rate constant” (K_(on)) andthe “off rate constant” (K_(off)) can be determined by calculation ofthe concentrations and the actual rates of association and dissociation.(See Nature 361:186-87 (1993)). The ratio of K_(off)/K_(on) enables thecancellation of all parameters not related to affinity, and is equal tothe dissociation constant K_(d). (See, generally, Davies et al. (1990)Annual Rev Biochem 59:439-473). An antibody of the present disclosure issaid to specifically bind to NaPi2b, when the equilibrium dissociationconstant (K_(d) or K_(D)) is ≤1 μM, preferably ≤100 nM, more preferably≤10 nM, and most preferably ≤100 pM to about 1 pM, as measured by assayssuch as radioligand binding assays or similar assays known to thoseskilled in the art.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the “isolated polynucleotide” (1)is not associated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (2) is operably linked toa polynucleotide which it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence. Polynucleotides inaccordance with the disclosure include nucleic acid molecules encodingthe heavy chain immunoglobulin molecules and portions thereofrepresented in SEQ ID NOs: 1, 3, 14, and 16, and nucleic acid moleculesencoding the light chain immunoglobulin molecules and portions thereofrepresented in SEQ ID NOs: 2, 4, 15, and 17.

The term “isolated protein” referred to herein means a protein of cDNA,recombinant RNA, or synthetic origin or some combination thereof, whichby virtue of its origin, or source of derivation, the “isolated protein”(1) is not associated with proteins found in nature, (2) is free ofother proteins from the same source, (3) is expressed by a cell from adifferent species, or (4) does not occur in nature.

The term “polypeptide” is used herein as a generic term to refer tonative protein, fragments, or analogs of a polypeptide sequence. Hence,native protein fragments, and analogs are species of the polypeptidegenus. Polypeptides in accordance with the disclosure comprise the heavychain immunoglobulin molecules and portions thereof presented in SEQ IDNOs: 1, 3, 14, and 16, and the light chain immunoglobulin molecules andportions thereof represented in SEQ ID NOs: 2, 4, 15, and 17 as well asantibody molecules formed by combinations comprising the heavy chainimmunoglobulin molecules with light chain immunoglobulin molecules, suchas kappa light chain immunoglobulin molecules, and vice versa, as wellas fragments and analogs thereof.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory orotherwise is naturally-occurring.

The term “operably linked” as used herein refers to positions ofcomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences.

The term “control sequence” as used herein refers to polynucleotidesequences that are necessary to effect the expression and processing ofcoding sequences to which they are ligated. The nature of such controlsequences differs depending upon the host organism in prokaryotes, suchcontrol sequences generally include promoter, ribosomal binding site,and transcription termination sequence in eukaryotes, generally, suchcontrol sequences include promoters and transcription terminationsequence. The term “control sequences” is intended to include, at aminimum, all components whose presence is essential for expression andprocessing, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences. The term “polynucleotide” as referred to herein means apolymeric boron of nucleotides of at least 10 bases in length, eitherribonucleotides or deoxyribonucleotides or a modified form of eithertype of nucleotide. The term includes single and double stranded formsof DNA.

The term “oligonucleotide” referred to herein includes naturallyoccurring, and modified nucleotides linked together by naturallyoccurring, and non-naturally occurring oligonucleotide linkages.Oligonucleotides are a polynucleotide subset generally comprising alength of 200 bases or fewer. Preferably oligonucleotides are 10 to 60bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or20 to 40 bases in length. Oligonucleotides are usually single stranded,e.g., for probes, although oligonucleotides may be double stranded,e.g., for use in the construction of a gene mutant. Oligonucleotidesdisclosed herein are either sense or antisense oligonucleotides.

The term “naturally occurring nucleotides” referred to herein includesdeoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” referred to herein includes nucleotides with modified orsubstituted sugar groups and the like. The term “oligonucleotidelinkages” referred to herein includes Oligonucleotides linkages such asphosphorothioate, phosphorodithioate, phosphoroselerloate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoroamidate, and the like. See e.g., LaPlanche et al. Nucl. AcidsRes. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984),Stein et al. Nucl. Acids Res. 16:3209 (1988), Zon et al. Anti CancerDrug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: APractical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford UniversityPress, Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510;Uhlmann and Peyman Chemical Reviews 90:543 (1990). An oligonucleotidecan include a label for detection, if desired.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotide or amino acid sequences: “referencesequence”, “comparison window”, “sequence identity”, “percentage ofsequence identity”, and “substantial identity”. A “reference sequence”is a defined sequence used as a basis for a sequence comparison areference sequence may be a subset of a larger sequence, for example, asa segment of a full-length cDNA or gene sequence given in a sequencelisting or may comprise a complete cDNA or gene sequence. Generally, areference sequence is at least 18 nucleotides or 6 amino acids inlength, frequently at least 24 nucleotides or 8 amino acids in length,and often at least 48 nucleotides or 16 amino acids in length. Since twopolynucleotides or amino acid sequences may each (1) comprise a sequence(i.e., a portion of the complete polynucleotide or amino acid sequence)that is similar between the two molecules, and (2) may further comprisea sequence that is divergent between the two polynucleotides or aminoacid sequences, sequence comparisons between two (or more) molecules aretypically performed by comparing sequences of the two molecules over a“comparison window” to identify and compare local regions of sequencesimilarity. A “comparison window”, as used herein, refers to aconceptual segment of at least 18 contiguous nucleotide positions or 6amino acids wherein a polynucleotide sequence or amino acid sequence maybe compared to a reference sequence of at least 18 contiguousnucleotides or 6 amino acid sequences and wherein the portion of thepolynucleotide sequence in the comparison window may comprise additions,deletions, substitutions, and the like (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2:482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.)85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage Release 7.0, (Genetics Computer Group, 575 Science Dr., Madison,Wis.), Geneworks, or MacVector software packages), or by inspection, andthe best alignment (i.e., resulting in the highest percentage ofhomology over the comparison window) generated by the various methods isselected.

The term “sequence identity” means that two polynucleotide or amino acidsequences are identical (i.e., on a nucleotide-by-nucleotide orresidue-by-residue basis) over the comparison window. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U or I) or residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the comparison window (i.e., the windowsize), and multiplying the result by 100 to yield the percentage ofsequence identity. The terms “substantial identity” as used hereindenotes a characteristic of a polynucleotide or amino acid sequence,wherein the polynucleotide or amino acid comprises a sequence that hasat least 85 percent sequence identity, preferably at least 90 to 95percent sequence identity, more usually at least 99 percent sequenceidentity as compared to a reference sequence over a comparison window ofat least 18 nucleotide (6 amino acid) positions, frequently over awindow of at least 24-48 nucleotide (8-16 amino acid) positions, whereinthe percentage of sequence identity is calculated by comparing thereference sequence to the sequence which may include deletions oradditions which total 20 percent or less of the reference sequence overthe comparison window. The reference sequence may be a subset of alarger sequence.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Green, Eds., Sinauer Associates,Sunderland? Mass. (1991)). Stereoisomers (e.g., D-amino acids) of thetwenty conventional amino acids, unnatural amino acids such as α-,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and otherunconventional amino acids may also be suitable components forpolypeptides of the present disclosure. Examples of unconventional aminoacids include: 4 hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,σ-N-methylarginine, and other similar amino acids and imino acids (e.g.,4-hydroxyproline). In the polypeptide notation used herein, theleft-hand direction is the amino terminal direction and the right-handdirection is the carboxy-terminal direction, in accordance with standardusage and convention.

Similarly, unless specified otherwise, the left-hand end ofsingle-stranded polynucleotide sequences is the 5′ end the left-handdirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction sequenceregions on the DNA strand having the same sequence as the RNA and whichare 5′ to the 5′ end of the RNA transcript are referred to as “upstreamsequences”, sequence regions on the DNA strand having the same sequenceas the RNA and which are 3′ to the 3′ end of the RNA transcript arereferred to as “downstream sequences”.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity, and mostpreferably at least 99 percent sequence identity.

Preferably, residue positions that are not identical differ byconservative amino acid substitutions.

Conservative amino acid substitutions refer to the interchangeability ofresidues having similar side chains. For example, a group of amino acidshaving aliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine valine,glutamic-aspartic, and asparagine-glutamine.

As discussed herein, minor variations in the amino acid sequences ofantibodies or immunoglobulin molecules are contemplated as beingencompassed by the present disclosure, providing that the variations inthe amino acid sequence maintain at least 75%, more preferably at least80%, 90%, 95%, and most preferably 99%. In particular, conservativeamino acid replacements are contemplated. Conservative replacements arethose that take place within a family of amino acids that are related intheir side chains. Genetically encoded amino acids are generally dividedinto families: (1) acidic amino acids are aspartate, glutamate; (2)basic amino acids are lysine, arginine, histidine; (3) non-polar aminoacids are alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan, and (4) uncharged polar amino acids are glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine. Thehydrophilic amino acids include arginine, asparagine, aspartate,glutamine, glutamate, histidine, lysine, serine, and threonine. Thehydrophobic amino acids include alanine, cysteine, isoleucine, leucine,methionine, phenylalanine, proline, tryptophan, tyrosine and valine.Other families of amino acids include (i) serine and threonine, whichare the aliphatic-hydroxy family; (ii) asparagine and glutamine, whichare the amide containing family; (iii) alanine, valine, leucine andisoleucine, which are the aliphatic family; and (iv) phenylalanine,tryptophan, and tyrosine, which are the aromatic family. For example, itis reasonable to expect that an isolated replacement of a leucine withan isoleucine or valine, an aspartate with a glutamate, a threonine witha serine, or a similar replacement of an amino acid with a structurallyrelated amino acid will not have a major effect on the binding orproperties of the resulting molecule, especially if the replacement doesnot involve an amino acid within a framework site. Whether an amino acidchange results in a functional peptide can readily be determined byassaying the specific activity of the polypeptide derivative. Assays aredescribed in detail herein. Analogs of antibodies or immunoglobulinmolecules can be readily prepared by those of ordinary skill in the art.Preferred amino- and carboxy-termini of analogs occur near boundaries offunctional domains. Structural and functional domains can be identifiedby comparison of the nucleotide and/or amino acid sequence data topublic or proprietary sequence databases. Preferably, computerizedcomparison methods are used to identify sequence motifs or predictedprotein conformation domains that occur in other proteins of knownstructure and/or function. Methods to identify protein sequences thatfold into a known three-dimensional structure are known. Bowie et al.Science 253:164 (1991). Thus, the foregoing examples demonstrate thatthose of skill in the art can recognize sequence motifs and structuralconformations that may be used to define structural and functionaldomains in accordance with the disclosure.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmuteins of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions(preferably conservative amino acid substitutions) may be made in thenaturally-occurring sequence (preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et at. Nature 354:105 (1991).

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcalorimetric methods). In certain situations, the label or marker canalso be therapeutic. Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent,biotinyl groups, predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). In someembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance. The term “pharmaceutical agent ordrug” as used herein refers to a chemical compound or compositioncapable of inducing a desired therapeutic effect when properlyadministered to a patient.

Other chemistry terms herein are used according to conventional usage inthe art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present.

Generally, a substantially pure composition will comprise more thanabout 80 percent of all macromolecular species present in thecomposition, more preferably more than about 85%, 90%, 95%, and 99%.Most preferably, the object species is purified to essential homogeneity(contaminant species cannot be detected in the composition byconventional detection methods) wherein the composition consistsessentially of a single macromolecular species.

The use of the articles “a”, “an”, and “the” in both the followingdescription and claims are to be construed to cover both the singularand the plural, unless otherwise indicated herein or clearlycontradicted by context. The terms “comprising”, “having”, “being of” asin “being of a chemical formula”, “including”, and “containing” are tobe construed as open terms (i.e., meaning “including but not limitedto”) unless otherwise noted. For example, a polymeric scaffold of acertain formula includes all the monomer units shown in the formula andmay also include additional monomer units not shown in the formula.Additionally whenever “comprising” or another open-ended term is used inan embodiment, it is to be understood that the same embodiment can bemore narrowly claimed using the intermediate term “consistingessentially of” or the closed term “consisting of.”

The term “about”, “approximately”, or “approximate”, when used inconnection with a numerical value, means that a collection or range ofvalues is included. For example, “about X” includes a range of valuesthat are ±20%, ±10%, ±5%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1% of X, where Xis a numerical value. In one embodiment, the term “about” refers to arange of values which are 5% more or less than the specified value. Inanother embodiment, the term “about” refers to a range of values whichare 2% more or less than the specified value. In another embodiment, theterm “about” refers to a range of values which are 1% more or less thanthe specified value.

Recitation of ranges of values are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. A range used herein, unless otherwisespecified, includes the two limits of the range. For example, theexpressions “x being an integer between 1 and 6” and “x being an integerof 1 to 6” both mean “x being 1, 2, 3, 4, 5, or 6”, i.e., the terms“between X and Y” and “range from X to Y, are inclusive of X and Y andthe integers there between.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to better illustrate thedisclosure and is not to be construed as a limitation on the scope ofthe claims unless explicitly otherwise claimed. No language in thespecification is to be construed as indicating that any non-claimedelement is essential to what is claimed.

“Biocompatible” as used herein is intended to describe compounds thatexert minimal destructive or host response effects while in contact withbody fluids or living cells or tissues. Thus a biocompatible group, asused herein, refers to an aliphatic, cycloalkyl, heteroaliphatic,heterocycloalkyl, aryl, or heteroaryl moiety, which falls within thedefinition of the term biocompatible, as defined above and herein. Theterm “Biocompatibility” as used herein, is also taken to mean that thecompounds exhibit minimal interactions with recognition proteins, e.g.,naturally occurring antibodies, cell proteins, cells and othercomponents of biological systems, unless such interactions arespecifically desirable. Thus, substances and functional groupsspecifically intended to cause the above minimal interactions, e.g.,drugs and prodrugs, are considered to be biocompatible. Preferably (withexception of compounds intended to be cytotoxic, such as, e.g.,antineoplastic agents), compounds are “biocompatible” if their additionto normal cells in vitro, at concentrations similar to the intendedsystemic in vivo concentrations, results in less than or equal to 1%cell death during the time equivalent to the half-life of the compoundin vivo (e.g., the period of time required for 50% of the compoundadministered in vivo to be eliminated/cleared), and their administrationin vivo induces minimal and medically acceptable inflammation, foreignbody reaction, immunotoxicity, chemical toxicity and/or other suchadverse effects. In the above sentence, the term “normal cells” refersto cells that are not intended to be destroyed or otherwisesignificantly affected by the compound being tested.

“Biodegradable”: As used herein, “biodegradable” polymers are polymersthat are susceptible to biological processing in vivo. As used herein,“biodegradable” compounds or moieties are those that, when taken up bycells, can be broken down by the lysosomal or other chemical machineryor by hydrolysis into components that the cells can either reuse ordispose of without significant toxic effect on the cells. The term“biocleavable” as used herein has the same meaning of “biodegradable”.The degradation fragments preferably induce little or no organ or celloverload or pathological processes caused by such overload or otheradverse effects in vivo. Examples of biodegradation processes includeenzymatic and non-enzymatic hydrolysis, oxidation and reduction.Suitable conditions for non-enzymatic hydrolysis of the biodegradableprotein-polymer-drug conjugates (or their components, e.g., thebiodegradable polymeric carrier and the linkers between the carrier andthe antibody or the drug molecule) described herein, for example,include exposure of the biodegradable conjugates to water at atemperature and a pH of lysosomal intracellular compartment.Biodegradation of some protein-polymer-drug conjugates (or theircomponents, e.g., the biodegradable polymeric carrier and the linkersbetween the carrier and the antibody or the drug molecule), can also beenhanced extracellularly, e.g., in low pH regions of the animal body,e.g., an inflamed area, in the close vicinity of activated macrophagesor other cells releasing degradation facilitating factors. In certainpreferred embodiments, the effective size of the polymer carrier atpH-7.5 does not detectably change over 1 to 7 days, and remains within50% of the original polymer size for at least several weeks. At pH-5, onthe other hand, the polymer carrier preferably detectably degrades over1 to 5 days, and is completely transformed into low molecular weightfragments within a two-week to several-month time frame. Polymerintegrity in such tests can be measured, for example, by size exclusionHPLC. Although faster degradation may be in some cases preferable, ingeneral it may be more desirable that the polymer degrades in cells withthe rate that does not exceed the rate of metabolization or excretion ofpolymer fragments by the cells. In preferred embodiments, the polymersand polymer biodegradation byproducts are biocompatible.

“Maleimido blocking compound”: as used herein refers to a compound thatcan react with maleimide to convert it to succinimide and “maleimidoblocking moiety” refers to the chemical moiety attached to thesuccinimide upon conversion. In certain embodiments, the maleimidoblocking compound is a compound having a terminal thiol group forreacting with the maleimide. In one embodiment, the maleimido blockingcompound is cysteine, N-acetyl cysteine, cysteine methyl ester, N-methylcysteine, 2-mercaptoethanol, 3-mercaptopropanoic acid, 2-mercaptoaceticacid, mercaptomethanol (i.e., HOCH₂SH), benzyl thiol in which phenyl issubstituted with one or more hydrophilic substituents, or3-aminopropane-1-thiol.

“Hydrophilic”: The term “hydrophilic” as it relates to substituents,e.g., on the polymer monomeric units or on a maleimido blocking moietyto render them hydrophilic or water soluble, does not essentially differfrom the common meaning of this term in the art, and denotes chemicalmoieties which contain ionizable, polar, or polarizable atoms, or whichotherwise may be solvated by water molecules. Thus a hydrophilic group,as used herein, refers to an aliphatic, cycloalkyl, heteroaliphatic,heterocycloalkyl, aryl or heteroaryl moiety, which falls within thedefinition of the term hydrophilic, as defined above. Examples ofparticular hydrophilic organic moieties which are suitable include,without limitation, aliphatic or heteroaliphatic groups comprising achain of atoms in a range of between about one and twelve atoms,hydroxyl, hydroxyalkyl, amine, carboxyl, amide, carboxylic ester,thioester, aldehyde, nitryl, isonitryl, nitroso, hydroxylamine,mercaptoalkyl, heterocycle, carbamates, carboxylic acids and theirsalts, sulfonic acids and their salts, sulfonic acid esters, phosphoricacids and their salts, phosphate esters, polyglycol ethers, polyamines,polycarboxylates, polyesters and polythioesters. In certain embodiments,hydrophilic substituents comprise a carboxyl group (COOH), an aldehydegroup (CHO), a ketone group (COC₁₋₄ alkyl), a methylol (CH₂OH) or aglycol (for example, CHOH—CH₂OH or CH—(CH₂OH)₂), NH₂, F, cyano, SO₃H,POSH, and the like.

The term “hydrophilic” as it relates to the polymers disclosed hereingenerally does not differ from usage of this term in the art, anddenotes polymers comprising hydrophilic functional groups as definedabove. In a preferred embodiment, hydrophilic polymer is a water-solublepolymer. Hydrophilicity of the polymer can be directly measured throughdetermination of hydration energy, or determined through investigationbetween two liquid phases, or by chromatography on solid phases withknown hydrophobicity, such as, for example, C4 or C18.

“Polymeric Carrier”: The term polymeric carrier, as used herein, refersto a polymer or a modified polymer, which is suitable for covalentlyattaching to or can be covalently attached to one or more drug moleculeswith a designated linker and/or one or more PBRMs with a designatedlinker.

“Physiological conditions”: The phrase “physiological conditions”, asused herein, relates to the range of chemical (e.g., pH, ionic strength)and biochemical (e.g., enzyme concentrations) conditions likely to beencountered in the extracellular fluids of living tissues. For mostnormal tissues, the physiological pH ranges from about 7.0 to 7.4.Circulating blood plasma and normal interstitial liquid representtypical examples of normal physiological conditions.

“Drug”: As used herein, the term “drug” refers to a compound which isbiologically active and provides a desired physiological effectfollowing administration to a subject in need thereof (e.g., an activepharmaceutical ingredient).

“Anti-angiogenesis agent” or “angiogenesis inhibitor” refers to a smallmolecular weight substance, a polynucleotide, a polypeptide, an isolatedprotein, a recombinant protein, an antibody, or conjugates or fusionproteins thereof, that inhibits angiogenesis, vasculogenesis, orundesirable vascular permeability, either directly or indirectly. Theangiogenesis inhibitor includes those agents that bind and block theangiogenic activity of the angiogenic factor or its receptor. Forexample, an anti-angiogenesis agent is an antibody or other antagonistto an angiogenic agent as including, but not limited to, antibodies toVEGF-A or to the VEGF-A receptor (e.g., KDR receptor or Flt-1 receptor),VEGF-trap, anti-PDGFR inhibitors such as Gleevec™ (Imatinib Mesylate).Anti-angiogenesis agents also include native angiogenesis inhibitors,e.g., angiostatin, endostatin, and the like.

“Cytotoxic”: As used herein the term “cytotoxic” means toxic to cells ora selected cell population (e.g., cancer cells). The toxic effect mayresult in cell death and/or lysis. In certain instances, the toxiceffect may be a sublethal destructive effect on the cell, e.g., slowingor arresting cell growth. In order to achieve a cytotoxic effect, thedrug or prodrug may be selected from a group consisting of a DNAdamaging agent, a microtubule disrupting agent, or a cytotoxic proteinor polypeptide, amongst others.

“Cytostatic”: As used herein the term “cytostatic” refers to a drug orother compound that inhibits or stops cell growth and/or multiplication.

“Small molecule”: As used herein, the term “small molecule” refers tomolecules, whether naturally-occurring or artificially created (e.g.,via chemical synthesis) that have a relatively low molecular weight.Preferred small molecules are biologically active in that they produce alocal or systemic effect in animals, preferably mammals, more preferablyhumans. In certain preferred embodiments, the small molecule is a drugand the small molecule is referred to as “drug molecule” or “drug” or“therapeutic agent”. In certain embodiments, the drug molecule has MWless than or equal to about 5 kDa. In other embodiments, the drugmolecule has MW less than or equal to about 1.5 kDa. In embodiments, thedrug molecule is selected from vinca alkaloids, auristatins,duocarmycins, tubulysins, non-natural camptothecin compounds,topoisomerase inhibitors, DNA binding drugs, kinase inhibitors, MEKinhibitors, KSP inhibitors, calicheamicins, SN38,pyrrolobenzodiazepines, and analogs thereof. Preferably, though notnecessarily, the drug is one that has already been deemed safe andeffective for use by an appropriate governmental agency or body, e.g.,the FDA. For example, drugs for human use listed by the FDA under 21C.F.R. §§ 330.5, 331 through 361, and 440 through 460; drugs forveterinary use listed by the FDA under 21 C.F.R. §§ 500 through 589,incorporated herein by reference, are all considered suitable for usewith the present hydrophilic polymers.

“Drug derivative” or “modified drug” or the like as used herein, refersto a compound that comprises the drug molecule intended to be deliveredby the conjugate disclosed herein and a functional group capable ofattaching the drug molecule to the polymeric carrier.

“Active form” as used herein refers to a form of a compound thatexhibits intended pharmaceutical efficacy in vivo or in vitro. Inparticular, when a drug molecule intended to be delivered by theconjugate disclosed herein is released from the conjugate, the activeform can be the drug itself or its derivatives, which exhibit theintended therapeutic properties. The release of the drug from theconjugate can be achieved by cleavage of a biodegradable bond of thelinker that attaches the drug to the polymeric carrier. The active drugderivatives accordingly can comprise a portion of the linker.

“PHF” refers to poly(l-hydroxymethylethylene hydroxymethyl-formal).

As used herein, the terms “polymer unit”, “monomeric unit”, “monomer”,“monomer unit”, “unit” all refer to a repeatable structural unit in apolymer.

As used herein, “molecular weight” or “MW” of a polymer or polymericcarrier/scaffold or polymer conjugates refers to the weight averagemolecular weight of the unmodified polymer unless otherwise specified.

“Immune checkpoint inhibitor” or “immune checkpoint inhibiting agent” or“immune checkpoint blocking agent” or “immune checkpoint modulator” asused herein, refers to an agent that binds an inhibitory immunecheckpoint protein and blocks its activity thereby enabling the immunesystem to recognize tumor cells and allowing a sustained immunotherapyresponse. The inhibition can be competitive or non-competitiveinhibition that can be steric or allosteric. In cases where an immunecheckpoint protein is an immune stimulating protein, an immunecheckpoint inhibitor acts to promote the activity of the immunestimulating protein, such as by binding and activating the stimulatoryimmune checkpoint protein or by inhibiting by interfering with, such asby binding or deactivating, inhibitors of the stimulatory immunecheckpoint protein. An example of an immune checkpoint inhibitor is ananti-immune checkpoint protein antibody.

“Immune checkpoints” as used herein refer to inhibitory pathways of theimmune system that are responsible for maintaining self-tolerance andmodulating the duration and amplitude of physiological immune responsesin peripheral tissues in order to minimize collateral tissue damage.Immune checkpoints are regulated by immune checkpoint proteins.

“Immune checkpoint protein” as used herein, refers to is a protein,typically a receptor (e.g., CTLA4 or PD-1) or a ligand (e.g., PD-L1)that regulates or modulates the extent of an immune response. The immunecheckpoint proteins can be inhibitory or stimulatory. In particular, theimmune checkpoint proteins are inhibitory to the activation of theimmune response. Thus, inhibition of an inhibitory immune checkpointprotein acts to stimulate or activate an immune response, such as T cellactivation and proliferation.

A “target” of an immune checkpoint inhibitor as used herein, is theimmune checkpoint protein to which the immune checkpoint inhibitor orimmune checkpoint inhibiting agent binds to block activity. Typically,the immune checkpoint inhibitor specifically binds to the target. Forexample, the target of the exemplary anti-CTLA4 antibody designatedipilimumab is CTLA4.

“Combination Therapy” as used herein refers to a treatment in which asubject is given two or more therapeutic agents, such as at least two orat least three therapeutic agents, for treating a single disease. Forpurposes herein, combination therapy includes therapy with aNaPi2b-targeted antibody-drug conjugate and an immune checkpointinhibitor.

As used herein “co-administration”, “co-administering” or“co-administered” refers to the administration of at least two differenttherapeutic agents sufficiently close in time. Such administration maybe done in any order, including simultaneous administration, as well astemporally spaced order from a few seconds up to several days apart.Such administration may also include more than a single administrationof one agent and/or independently the other agent. The administration ofthe agents may be by the same or different routes.

As used herein, “anti-CTLA4 antibody” refers to any antibody thatspecifically binds to cytotoxic T-lymphocyte-associated protein 4(CTLA4) or a soluble fragment thereof and blocks the binding of ligandsto CTLA4, thereby resulting in competitive inhibition of CTLA4 andinhibition of CTLA4-mediated inhibition of T cell activation. Hence,anti-CTLA4 antibodies are CTLA4 inhibitors. Reference to anti-CTLA4antibodies herein include a full-length antibody and derivativesthereof, such as antigen-binding fragments thereof that specificallybind to CTLA4. Exemplary anti-CTLA4 antibodies include, but are notlimited to, ipilimumab or tremelimumab, or a derivative orantigen-binding fragment thereof.

As used herein, a “cytotoxic T-lymphocyte-associated protein 4” (CTLA4;also known as CD 152) antigen refers to an inhibitory receptor of theimmunoglobulin superfamily, that is bound by ligands such as CD80 (alsocalled B7-1) and CD86, (also called B7-2). CTLA4 includes human andnon-human proteins. In particular, CTLA4 antigen includes human CTLA4,which has the sequence of amino acids set forth in SEQ ID NO: 10 (seee.g., GenBank Accession No. AAL07473.1).

As used herein, “anti-PD-1 antibody” refers to any antibody thatspecifically binds to programmed cell death protein 1 (PD-1) or asoluble fragment thereof and blocks the binding of ligands to PD-1,thereby resulting in competitive inhibition of PD-1 and inhibition ofPD-1 mediated inhibition of T cell activation. Hence, anti-PD-1antibodies are PD-1 inhibitors. Reference to anti-PD-1 antibodies hereininclude a full-length antibody and derivatives thereof, such asantigen-binding fragments thereof that specifically bind to PD-1.Exemplary anti-PD-1 antibodies include, but are not limited to,nivolumab, MK-3475, pidilizumab, or a derivative or antigen-bindingfragment thereof.

As used herein, a “programmed cell death protein 1” (PD-1) antigenrefers to an inhibitory receptor, that is a type 1 membrane protein andis bound by ligands such as PD-L1 and PD-L2, which are members of the B7family. PD-1 includes human and non-human proteins. In particular, PD-1antigen includes human PD-1, which has the sequence of amino acids setforth in SEQ ID NO: 299 (see e.g., UniProt Accession No. Q15116.3). Asused herein, anti-PD-L1 antibody refers to an antibody that specificallybinds to programed death-ligand 1 (PD-L1) or a soluble fragment thereofand blocking the binding of the ligand to PD-1, thereby resulting incompetitive inhibition of PD-1 and inhibition of PD-1 mediatedinhibition of T cell activity. Hence, anti-PD-LI antibodies are PD-1inhibitors. Reference to anti-PD-L1 antibodies herein include afull-length antibody and derivatives thereof, such as antigen-bindingfragments thereof that specifically bind to PD-L1. Exemplary anti-PD-L1antibodies include, but are not limited to, BMS-936559, MPDL3280A,MEDI4736 or a derivative or antigen-binding fragment thereof.

As used herein, “dosing regimen” or “dosage regimen” refers to theamount of agent, for example, the composition containing aNaPi2b-targeted antibody-drug conjugate, administered, and the frequencyof administration. The dosing regimen is a function of the disease orcondition to be treated, and thus can vary.

As used herein, “frequency” of administration refers to the time betweensuccessive administrations of treatment. For example, frequency can bedays, weeks or months. For example, frequency can be more than onceweekly, for example, twice a week, three times a week, four times aweek, five times a week, six times a week or daily. Frequency also canbe one, two, three or four weeks. The particular frequency is a functionof the particular disease or condition treated. Generally, frequency ismore than once weekly, and generally is twice weekly.

As used herein, a “cycle of administration” refers to the repeatedschedule of the dosing regimen of administration of the enzyme and/or asecond agent that is repeated over successiveadministrations. Forexample, an exemplary cycle of administration is a 28 day cycle withadministration twice weekly for three weeks, followed by one-week ofdiscontinued dosing.

As used herein, when referencing dosage based on mg/kg of the subject,an average human subject is considered to have a mass of about 70 kg-75kg, such as 70 kg and a body surface area (BSA) of 1.73 m. As usedherein, amelioration of the symptoms of a particular disease or disorderby a treatment, such as by administration of a pharmaceuticalcomposition or other therapeutic, refers to any lessening, whetherpermanent or temporary, lasting or transient, of the symptoms or,adverse effects of a condition, such as, for example, reduction ofadverse effects associated with or that occur upon administration of aNaPi2b-targeted antibody-drug conjugate.

As used herein, “treating” or “treat” describes the management and careof a patient for the purpose of combating a disease, condition, ordisorder and includes the administration of a conjugate of thedisclosure, or a pharmaceutical composition thereof, to alleviate thesymptoms or complications of a disease, condition or disorder, or toeliminate the disease, condition or disorder.

As used herein, “prevention” or “prophylaxis” refers to reduction in therisk of developing a disease or condition, or reduction or eliminationof the onset of the symptoms or complications of the disease, conditionor disorder.

The term “effective amount” or “sufficient amount”, as it refers to anactive agent, refers to the amount necessary to elicit the desiredbiological response. As used herein, a “therapeutically effectiveamount” or a “therapeutically effective dose” refers to an amount orquantity of an agent, compound, material, or composition containing acompound that is at least sufficient to produce a detectable therapeuticeffect. The effect can be detected by any assay method known in the art.The precise effective amount for a subject will depend upon thesubject's body weight, size, and health; the nature and extent of thecondition; and the therapeutic selected for administration.

A “subject” includes a mammal. The mammal can be e.g., any mammal, e.g.,a human, primate, bird, mouse, rat, fowl, dog, cat, cow, horse, goat,camel, sheep or a pig. Preferably, the mammal is a human.

As used herein, “unit dose form” or “unit dosage form” refers tophysically discrete units suitable for human and animal subjects andpackaged individually as is known in the art.

As used herein, a single dosage formulation refers to a formulation as asingle dose.

As used herein a “kit” refers to a combination of components, such as acombination of the compositions herein and another item for a purposeincluding, but not limited to, reconstitution, activation andinstruments/devices for delivery, administration, diagnosis andassessment of a biological activity or property. Kits optionally includeinstructions of use.

The present disclosure is intended to include all isotopes of atomsoccurring in the present compounds. Isotopes include those atoms havingthe same atomic number but different mass numbers. By way of generalexample and without limitation, isotopes of hydrogen include tritium anddeuterium. Isotopes of carbon include C-13 and C-14.

The present disclosure is intended to include all isomers of thecompound, which refers to and includes, optical isomers, and tautomericisomers, where optical isomers include enantiomers and diastereomers,chiral isomers and non-chiral isomers, and the optical isomers includeisolated optical isomers as well as mixtures of optical isomersincluding racemic and non-racemic mixtures; where an isomer may be inisolated form or in a mixture with one or more other isomers.

NaPi2b Antibodies

Antibody-drug conjugates disclosed herein include monoclonal antibodiesthat specifically recognize NaPi2b and have the ability to inhibitNaPi2b activity.

Exemplary antibodies used in the antibody-drug conjugates disclosedherein include, for example, the antibodies referred to herein as theXMT 1535 antibody and/or the 10H1.11.4B antibody. These antibodies showspecificity for human NaPi2b and they have been shown to inhibit thefunctional activity of NaPi2b in vitro.

Each of the NaPi2b monoclonal antibodies described herein includes aheavy chain (HC), heavy chain variable region (VH), light chain (LC),and a light chain variable region (VL), as shown in the amino acid andcorresponding nucleic acid sequences presented below. The variable heavychain region and variable light chain region for each antibody areshaded in the amino acid sequences below. The complementaritydetermining regions (CDRs) of the heavy chain and the light chain areunderlined in the amino acid sequences presented below. The amino acidsencompassing the complementarity determining regions (CDRs) for the XMT1535 antibody are as defined by E. A. Kabat et al. (See Kabat, E. A., etal., Sequences of Protein of immunological interest, Fifth Edition, USDepartment of Health and Human Services, US Government Printing Office(1991)) and are disclosed in U.S. Pat. No. 8,603,474, and the aminoacids encompassing the CDRs for the 10H1.11.4B antibody are as definedin U.S. Pat. No. 8,535,675.

>XMT 1535 Heavy Chain Amino Acid Sequence (Heavy chain variable region (SEQ ID NO: 3) + IgG1 Heavy chain constant region (SEQ ID NO: 11))  (SEQ ID NO: 1) QVQLVQSGAEVVKPGASVKMSCKAS GYTFTGYNIHWVKQAPGQGLEWIG AIYPGNGDTSYKQKFRG   RATLTADTSTSTVYMELSSLRSEDSAVYYCARGETARATFAY WGQGTLVTVSSGASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG*  (SEQ ID NO: 5)CDRH1: GYTFTGYNIH  (SEQ ID NO: 6) CDRH2: AIYPGNGDTSYKQKFRG (SEQ ID NO: 7)CDRH3: GETARATFAY  >XMT 1535 Light Chain Amino Acid Sequence (Light chain variable region (SEQ ID NO: 4) + Light chain constant region (SEQ ID  NO: 12)) (SEQ ID NO: 2) DIQMTQSPSSLSASVGDRVTITC SASQDIGNFLN WYQQKPGKTVKVLIYYTSSLYS GVPSRFSGSG  SGTDYTLTISSLQPEDFATYYC QQYSKLPLTFGQGTKLELKRRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC  (SEQ ID NO: 8) CDRL1: SASQDIGNFLN  (SEQ ID NO: 9)CDRL2: YTSSLYS  (SEQ ID NO: 10)CDRL3: QQYSKLPLT  >10H1.11.4B Heavy Chain Amino Acid Sequence (Heavy chain variable region (SEQ ID NO: 16) + IgG1 Heavy chain constant region (SEQ ID NO: 13))  (SEQ ID NO: 14) EVQLVESGGGLVQPGGSLRLSCAAS GFSFSDFAMSWVRQAPGKGLEWV ATIGRVAFHTYYPDSMKG   RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHRGFDVGHFDF WGQGTLVTVSSASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG*  (SEQ ID NO: 18)CDRH1: GFSFSDFAMS  (SEQ ID NO: 19) CDRH2: ATIGRVAFHTYYPDSMKG (SEQ ID NO: 20)CDRH3: ARHRGFDVGHFDF  >10H1.11.4B Light Chain Amino Acid Sequence (Light chain variable region (SEQ ID NO: 17) + Light chain constant region (SEQ ID  NO: 12)) (SEQ ID NO: 15) DIQMTQSPSSLSASVGDRVTITC RSSETLVHSSGNTYLE WYQQKPGKAPKLLIYRVSNRFS GVPSR  FSGSGSGTDFTLTISSLQPEDFATYYC FQGSFNPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKS GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC*  (SEQ ID NO: 21) CDRL1: RSSETLVHSSGNTYLE (SEQ ID NO: 22) CDRL2: RVSNRFS  (SEQ ID NO: 23) CDRL3: FQGSFNPLT 

Also included in the disclosure are antibodies that bind to the sameepitope or cross compete for binding to the same epitope as theantibodies described herein. For example, antibodies disclosed hereinspecifically bind to NaPi2b, wherein the antibody binds to an epitopethat includes one or more amino acid residues on human NaPi2b (e.g.,GenBank Accession No. 095436.3).

Antibodies disclosed herein specifically bind to an epitope on thefull-length human NaPi2b comprising the amino acid sequence:

(SEQ ID NO: 24)  1 MAPWPELGDA QPNPDKYLEG AAGQQPTAPD KSKETNKTDN TEAPVTKIEL  51 LPSYSTATLI DEPTEVDDPW NLPTLQDSGI KWSERDTKGK ILCFFQGIGR 101 LILLLGFLYF FVCSLDILSS AFQLVGGKMA GQFFSNSSIM SNPLLGLVIG 151 VLVTVLVQSS STSTSIVVSM VSSSLLTVRA AIPIIMGANI GTSITNTIVA 201 LMQVGDRSEF RRAFAGATVH DFFNWLSVLV LLPVEVATHY LEIITQLIVE 251 SFHFKNGEDA PDLLKVITKP FTKLIVQLDK KVISQIAMND EKAKNKSLVK 301 IWCKTFTNKT QINVTVPSTA NCTSPSLCWT DGIQNWTMKN VTYKENIAKC 351 QHIFVNFHLP DLAVGTILLI LSLLVLCGCL IMIVKILGSV LKGQVATVIK 401 KTINTDFPFP FAWLTGYLAI LVGAGMTFIV QSSSVFTSAL TPLIGIGVIT 451 IERAYPLTLG SNIGTTTTAI LAALASPGNA LRSSLQIALC HFFFNISGIL 501 LWYPIPFTRL PIRMAKGLGN ISAKYRWFAV FYLIIFFFLI PLTVFGLSLA 551 GWRVLVGVGV PVVFIIILVL CLRLLQSRCP RVLPKKLQNW NFLPLWMRSL 601 KPWDAVVSKF TGCFQMRCCC CCRVCCRACC LLCDCPKCCR CSKCCEDLEE 651 AQEGQDVPVK APETFDNITI SREAQGEVPA SDSKTECTAL 

Antibodies disclosed herein specifically bind to an epitope on anextracellular domain (ECD) of the human NaPi2b.

Those skilled in the art will recognize that it is possible todetermine, without undue experimentation, if a monoclonal antibody hasthe same specificity as a monoclonal antibody disclosed herein (e.g.,XMT 1535, 10H1.11.4B) by ascertaining whether the former prevents thelatter from binding to a natural binding partner or other molecule knownto be associated with NaPi2b. If the monoclonal antibody being testedcompetes with the monoclonal antibody disclosed herein, as shown by adecrease in binding by the monoclonal antibody disclosed herein, thenthe two monoclonal antibodies bind to the same, or a closely related,epitope.

An alternative method for determining whether a monoclonal antibody hasthe specificity of monoclonal antibody disclosed herein is topre-incubate the monoclonal antibody disclosed herein with solubleNaPi2b (with which it is normally reactive), and then add the monoclonalantibody being tested to determine if the monoclonal antibody beingtested is inhibited in its ability to bind NaPi2b. If the monoclonalantibody being tested is inhibited then, in all likelihood, it has thesame, or functionally equivalent, epitopic specificity as the monoclonalantibody disclosed herein.

Screening of monoclonal antibodies disclosed herein, can also be carriedout, e.g., by measuring NaPi2b-mediated activity, and determiningwhether the test monoclonal antibody is able to modulate, block,inhibit, reduce, antagonize, neutralize or otherwise interfere withNaPi2b activity.

NaPi2b antibodies are generated, for example, using various proceduresknown within the art may be used for the production of monoclonalantibodies directed against NaPi2b, or against derivatives, fragments,analogs homologs or orthologs thereof. (See, for example, Antibodies: ALaboratory Manual, Harlow E, and Lane D, 1988, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., incorporated herein byreference). Fully human antibodies are antibody molecules in which theentire sequence of both the light chain and the heavy chain, includingthe CDRs, arise from human genes. Such antibodies are termed “humanantibodies” or “fully human antibodies” herein. Human monoclonalantibodies are prepared, for example, using the procedures described inthe Examples provided below. Human monoclonal antibodies can be alsoprepared by using the trioma technique; the human B-cell hybridomatechnique (see Kozbor, et al., 1983 Immunol Today 4: 72); and the EBVhybridoma technique to produce human monoclonal antibodies (see Cole, etal., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss,Inc., pp. 77-96). Human monoclonal antibodies may be utilized and may beproduced by using human hybridomas (see Cote, et al., 1983. Proc NatlAcad Sci USA 80: 2026-2030) or by transforming human B-cells withEpstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).

Antibodies are purified by well-known techniques, such as affinitychromatography using protein A or protein G, which provide primarily theIgG fraction of immune serum. Subsequently, or alternatively, thespecific antigen that is the target of the immunoglobulin sought, or anepitope thereof, may be immobilized on a column to purify the immunespecific antibody by immunoaffinity chromatography. Purification ofimmunoglobulins is discussed, for example, by D. Wilkinson (TheScientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14,No. 8 (Apr. 17, 2000), pp. 25-28).

Monoclonal antibodies that modulate, block, inhibit, reduce, antagonize,neutralize or otherwise interfere with NaPi2b activity are generated,e.g., by immunizing an animal with membrane bound and/or soluble NaPi2b,such as, for example, murine, rat or human NaPi2b or an immunogenicfragment, derivative or variant thereof. Alternatively, the animal isimmunized with cells transfected with a vector containing a nucleic acidmolecule encoding NaPi2b such that NaPi2b is expressed and associatedwith the surface of the transfected cells. Alternatively, the antibodiesare obtained by screening a library that contains antibody or antigenbinding domain sequences for binding to NaPi2b. This library isprepared, e.g., in bacteriophage as protein or peptide fusions to abacteriophage coat protein that is expressed on the surface of assembledphage particles and the encoding DNA sequences contained within thephage particles (i.e., “phage displayed library”). Hybridomas resultingfrom myeloma/B cell fusions are then screened for reactivity to NaPi2b.Additionally, the antibodies by selected from, and optionally optimizedin, yeast antibody display libraries and yeast library presentationsystems as described in, e.g.: Blaise L, Wehnert A, Steukers M P, vanden Beucken T, Hoogenboom H R, Hufton S E. Construction anddiversification of yeast cell surface displayed libraries by yeastmating: application to the affinity maturation of Fab antibodyfragments. Gene. 2004 Nov. 24; 342(2):211-8; Boder E T, Wittrup K D.Yeast surface display for screening combinatorial polypeptide libraries.Nat Biotechnol. 1997 June; 15(6):553-7; Kuroda K, Ueda M. Cell surfaceengineering of yeast for applications in white biotechnology. BiotechnolLett. 2011 January; 33(1):1-9. doi: 10.1007/s10529-010-0403-9. Review;Lauer T M, Agrawal N J, Chennamsetty N, Egodage K, Helk B, Trout B L.Developability index: a rapid in silico tool for the screening ofantibody aggregation propensity. J Pharm Sci. 2012 January;101(1):102-15; Orcutt K. D. and Wittrup K. D. (2010), 207-233 doi:10.1007/978-3-642-01144-3_15; Rakestraw J A, Aird D, Aha P M, Baynes BM, Lipovsek D. Secretion-and-capture cell-surface display for selectionof target-binding proteins. Protein Eng Des Sel. 2011 June;24(6):525-30; U.S. Pat. Nos. 8,258,082; 6,300,064; 6,696,248; 6,165,718;6,500,644; 6,291,158; 6,291,159; 6,096,551; 6,368,805; 6,500,644.Exemplary yeast library presentation systems are described in, e.g.,WO2008118476; WO2009/036379; WO2010105256; and WO2012009568. In certainembodiments, such yeast antibody display libraries or yeast librarypresentation systems are designed to mimic or reflect the diversitycharacteristic of the human preimmune antibody repertoire. In certainembodiments such yeast antibody display library diversity or yeastlibrary presentation system diversity is generated in silico. In certainembodiments such yeast antibody display libraries or yeast librarypresentation systems comprise Saccharomyces yeast cells, such asSaccharomyces Cerevisiae cells. In certain embodiments such yeastantibody display libraries or yeast library presentation systemscomprise Pichia cells.

Monoclonal antibodies are prepared, for example, using hybridomamethods, such as those described by Kohler and Milstein, Nature, 256:495(1975). In a hybridoma method, a mouse, hamster, or other appropriatehost animal, is typically immunized with an immunizing agent to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the immunizing agent. Alternatively, thelymphocytes can be immunized in vitro.

The immunizing agent will typically include the protein antigen, afragment thereof or a fusion protein thereof. Generally, eitherperipheral blood lymphocytes are used if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986) pp. 59-103).Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells can becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (“HAT medium”), which substances prevent thegrowth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of monoclonalantibodies. (See Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63)).

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against theantigen. Preferably, the binding specificity of monoclonal antibodiesproduced by the hybridoma cells is determined by immunoprecipitation orby an in vitro binding assay, such as radioimmunoassay (MA) orenzyme-linked immunoabsorbent assay (ELISA). Such techniques and assaysare known in the art. The binding affinity of the monoclonal antibodycan, for example, be determined by the Scatchard analysis of Munson andPollard, Anal. Biochem., 107:220 (1980). Moreover, in therapeuticapplications of monoclonal antibodies, it is important to identifyantibodies having a high degree of specificity and a high bindingaffinity for the target antigen.

After the desired hybridoma cells are identified, the clones can besubcloned by limiting dilution procedures and grown by standard methods.(See Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103). Suitable culture media for this purposeinclude, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640medium. Alternatively, the hybridoma cells can be grown in vivo asascites in a mammal.

The monoclonal antibodies secreted by the subclones can be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxyapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

Monoclonal antibodies can also be made by recombinant DNA methods, suchas those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies disclosed herein can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cellsdisclosed herein serve as a preferred source of such DNA. Once isolated,the DNA can be placed into expression vectors, which are thentransfected into host cells such as simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. The DNA also can be modified, forexample, by substituting the coding sequence for human heavy and lightchain constant domains in place of the homologous murine sequences (seeU.S. Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. Such anon-immunoglobulin polypeptide can be substituted for the constantdomains of an antibody disclosed herein, or can be substituted for thevariable domains of one antigen-combining site of an antibody disclosedherein to create a chimeric bivalent antibody.

Monoclonal antibodies disclosed herein include fully human antibodies orhumanized antibodies. These antibodies are suitable for administrationto humans without engendering an immune response by the human againstthe administered immunoglobulin.

A humanized or fully human NaPi2b antibody is generated, for example,using the procedures described in the Examples provided below.

In other, alternative methods, a NaPi2b antibody is developed, forexample, using phage-display methods using antibodies containing onlyhuman sequences. Such approaches are well-known in the art, e.g., inWO92/01047 and U.S. Pat. No. 6,521,404, which are hereby incorporated byreference. In this approach, a combinatorial library of phage carryingrandom pairs of light and heavy chains are screened using natural orrecombinant source of NaPi2b. In another approach, a NaPi2b antibody canbe produced by a process wherein at least one step of the processincludes immunizing a transgenic, non-human animal with human NaPi2bprotein. In this approach, some of the endogenous heavy and/or kappalight chain loci of this xenogeneic non-human animal have been disabledand are incapable of the rearrangement required to generate genesencoding immunoglobulins in response to an antigen. In addition, atleast one human heavy chain locus and at least one human light chainlocus have been stably transfected into the animal. Thus, in response toan administered antigen, the human loci rearrange to provide genesencoding human variable regions immunospecific for the antigen. Uponimmunization, therefore, the XenoMouse produces B-cells that secretefully human immunoglobulins.

A variety of techniques are well-known in the art for producingxenogeneic non-human animals. For example, see U.S. Pat. Nos. 6,075,181and 6,150,584, which is hereby incorporated by reference in itsentirety. This general strategy was demonstrated in connection withgeneration of the first XenoMouse™ strains as published in 1994. SeeGreen et al. Nature Genetics 7:13-21 (1994), which is herebyincorporated by reference in its entirety. See also, U.S. Pat. Nos.6,162,963, 6,150,584, 6,114,598, 6,075,181, and 5,939,598 and JapanesePatent Nos. 3 068 180 B2, 3 068 506 B2, and 3 068 507 B2 and EuropeanPatent No., EP 0 463 151 B1 and International Patent Applications No. WO94/02602, WO 96/34096, WO 98/24893, WO 00/76310 and related familymembers.

In an alternative approach, others have utilized a “minilocus” approachin which an exogenous Ig locus is mimicked through the inclusion ofpieces (individual genes) from the Ig locus. Thus, one or more VH genes,one or more DH genes, one or more JH genes, a mu constant region, and asecond constant region (preferably a gamma constant region) are formedinto a construct for insertion into an animal. See e.g., U.S. Pat. Nos.5,545,806; 5,545,807; 5,591,669; 5,612,205; 5,625,825; 5,625,126;5,633,425; 5,643,763; 5,661,016; 5,721,367; 5,770,429; 5,789,215;5,789,650; 5,814,318; 5,877; 397; 5,874,299; 6,023,010; and 6,255,458;and European Patent No. 0 546 073 B1; and International PatentApplication Nos. WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO98/24884 and related family members.

Generation of human antibodies from mice in which, through microcellfusion, large pieces of chromosomes, or entire chromosomes, have beenintroduced, has also been demonstrated. See European Patent ApplicationNos. 773 288 and 843 961.

Human anti-mouse antibody (HAMA) responses have led the industry toprepare chimeric or otherwise humanized antibodies. While chimericantibodies have a human constant region and an immune variable region,it is expected that certain human anti-chimeric antibody (HACA)responses will be observed, particularly in chronic or multi-doseutilizations of the antibody. Thus, it would be desirable to providefully human antibodies against NaPi2b in order to vitiate or otherwisemitigate concerns and/or effects of HAMA or HACA response.

The production of antibodies with reduced immunogenicity is alsoaccomplished via humanization, chimerization and display techniquesusing appropriate libraries. It will be appreciated that murineantibodies or antibodies from other species can be humanized orprimatized using techniques well known in the art. See e.g., Winter andHarris Immunol Today 14:43 46 (1993) and Wright et al. Crit, Reviews inImmunol. 12125-168 (1992). The antibody of interest may be engineered byrecombinant DNA techniques to substitute the CH₁, CH₂, CH₃, hingedomains, and/or the framework domain with the corresponding humansequence (See WO 92102190 and U.S. Pat. Nos. 5,530,101; 5,585,089;5,693,761; 5,693,792; 5,714,350; and 5,777,085). Also, the use of IgcDNA for construction of chimeric immunoglobulin genes is known in theart (Liu et al. P.N.A.S. 84:3439 (1987) and J. Immunol. 139:3521(1987)). mRNA is isolated from a hybridoma or other cell producing theantibody and used to produce cDNA. The cDNA of interest may be amplifiedby the polymerase chain reaction using specific primers (U.S. Pat. Nos.4,683,195 and 4,683,202). Alternatively, a library is made and screenedto isolate the sequence of interest. The DNA sequence encoding thevariable region of the antibody is then fused to human constant regionsequences. The sequences of human constant regions genes may be found inKabat et al. (1991) Sequences of Proteins of immunological Interest,N.I.H. publication no. 91-3242. Human C region genes are readilyavailable from known clones. The choice of isotype will be guided by thedesired effecter functions, such as complement fixation, or activity inantibody-dependent cellular cytotoxicity. Preferred isotypes are IgG1,IgG3 and IgG4. Either of the human light chain constant regions, kappaor lambda, may be used. The chimeric, humanized antibody is thenexpressed by conventional methods.

Further, human antibodies or antibodies from other species can begenerated through display type technologies, including, withoutlimitation, phage display, retroviral display, ribosomal display, andother techniques, using techniques well known in the art and theresulting molecules can be subjected to additional maturation, such asaffinity maturation, as such techniques are well known in the art.Wright et al. Crit, Reviews in Immunol. 12125-168 (1992), Hanes andPluckthun PNAS USA 94:4937-4942 (1997) (ribosomal display), Parmley andSmith Gene 73:305-318 (1988) (phage display), Scott, TIBS, vol.17:241-245 (1992), Cwirla et al. PNAS USA 87:6378-6382 (1990), Russel etal. Nucl. Acids Research 21:1081-1085 (1993), Hoganboom et al. Immunol.Reviews 130:43-68 (1992), Chiswell and McCafferty TIBTECH; 10:80-8A(1992), and U.S. Pat. No. 5,733,743. If display technologies areutilized to produce antibodies that are not human, such antibodies canbe humanized as described above.

Using these techniques, antibodies can be generated to NaPi2b expressingcells, soluble forms of NaPi2b, epitopes or peptides thereof, andexpression libraries thereto (See e.g., U.S. Pat. No. 5,703,057) whichcan thereafter be screened as described above for the activitiesdescribed herein.

The NaPi2b antibodies disclosed herein can be expressed by a vectorcontaining a DNA segment encoding the single chain antibody describedabove.

These can include vectors, liposomes, naked DNA, adjuvant-assisted DNA,gene gun, catheters, etc. Vectors include chemical conjugates such asdescribed in WO 93/64701, which has targeting moiety (e.g., a ligand toa cellular surface receptor), and a nucleic acid binding moiety (e.g.,polylysine), viral vector (e.g., a DNA or RNA viral vector), fusionproteins such as described in PCT/US 95/02140 (WO 95/22618) which is afusion protein containing a target moiety (e.g., an antibody specificfor a target cell) and a nucleic acid binding moiety (e.g., aprotamine), plasmids, phage, etc. The vectors can be chromosomal,non-chromosomal or synthetic.

Preferred vectors include viral vectors, fusion proteins and chemicalconjugates. Retroviral vectors include moloney murine leukemia viruses.DNA viral vectors are preferred. These vectors include pox vectors suchas orthopox or avipox vectors, herpesvirus vectors such as a herpessimplex I virus (HSV) vector (see Geller, A. I. et al., J. Neurochem,64:487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D.Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A. I.et al., Proc Natl. Acad. Sci.: U.S.A. 90:7603 (1993); Geller, A. I., etal., Proc Natl. Acad. Sci USA 87:1149 (1990), Adenovirus Vectors (seeLeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat.Genet 3:219 (1993); Yang, et al., J. Virol. 69:2004 (1995) andAdeno-associated Virus Vectors (see Kaplitt, M. G. et al., Nat. Genet.8:148 (1994).

Pox viral vectors introduce the gene into the cells cytoplasm. Avipoxvirus vectors result in only a short term expression of the nucleicacid. Adenovirus vectors, adeno-associated virus vectors and herpessimplex virus (HSV) vectors are preferred for introducing the nucleicacid into neural cells. The adenovirus vector results in a shorter termexpression (about 2 months) than adeno-associated virus (about 4months), which in turn is shorter than HSV vectors. The particularvector chosen will depend upon the target cell and the condition beingtreated. The introduction can be by standard techniques, e.g.,infection, transfection, transduction or transformation. Examples ofmodes of gene transfer include e.g., naked DNA, CaPO4 precipitation,DEAE dextran, electroporation, protoplast fusion, lipofection, cellmicroinjection, and viral vectors.

The vector can be employed to target essentially any desired targetcell. For example, stereotaxic injection can be used to direct thevectors (e.g., adenovirus, HSV) to a desired location. Additionally, theparticles can be delivered by intracerebroventricular (icy) infusionusing a minipump infusion system, such as a SynchroMed Infusion System.A method based on bulk flow, termed convection, has also proveneffective at delivering large molecules to extended areas of the brainand may be useful in delivering the vector to the target cell. (See Boboet al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al.,Am. J. Physiol. 266:292-305 (1994)). Other methods that can be usedinclude catheters, intravenous, parenteral, intraperitoneal andsubcutaneous injection, and oral or other known routes ofadministration.

These vectors can be used to express large quantities of antibodies thatcan be used in a variety of ways. For example, to detect the presence ofNaPi2b in a sample. The antibody can also be used to try to bind to anddisrupt NaPi2b-related signaling.

NaPi2b-targeted Antibody Conjugates:

The disclosure also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a toxin (e.g., an enzymaticallyactive toxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a radioactive isotope (i.e., a radioconjugate).

Enzymatically active toxins and fragments thereof that can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolacca americana proteins (PAPI, PAPII,and PAP-S), Momordica charantia inhibitor, curcin, crotin, Saponariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the trichothecenes. A variety of radionuclides areavailable for the production of radioconjugated antibodies. Examplesinclude ¹²²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolylene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. (See WO94/11026).

Those of ordinary skill in the art will recognize that a large varietyof possible moieties can be coupled to the resultant antibodiesdisclosed herein. (See, for example, “Conjugate Vaccines”, Contributionsto Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr (eds),Carger Press, New York, (1989), the entire contents of which areincorporated herein by reference).

Coupling may be accomplished by any chemical reaction that will bind thetwo molecules so long as the antibody and the other moiety retain theirrespective activities. This linkage can include many chemicalmechanisms, for instance covalent binding, affinity binding,intercalation, coordinate binding and complexation. The preferredbinding is, however, covalent binding. Covalent binding can be achievedeither by direct condensation of existing side chains or by theincorporation of external bridging molecules. Many bivalent orpolyvalent linking agents are useful in coupling protein molecules, suchas the antibodies of the present disclosure, to other molecules. Forexample, representative coupling agents can include organic compoundssuch as thioesters, carbodiimides, succinimide esters, diisocyanates,glutaraldehyde, diazobenzenes and hexamethylene diamines. This listingis not intended to be exhaustive of the various classes of couplingagents known in the art but, rather, is exemplary of the more commoncoupling agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549(1984); Jansen et al., Immunological Reviews 62:185-216 (1982); andVitetta et al., Science 238:1098 (1987).

Preferred linkers are described in the literature. (See, for example,Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describing use ofMBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, U.S. Pat.No. 5,030,719, describing use of halogenated acetyl hydrazide derivativecoupled to an antibody by way of an oligopeptide linker. Particularlypreferred linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride; (ii) SMPT(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio)-toluene(Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio) propionamido]hexanoate (Pierce Chem. Co., Cat.#21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat.#2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem.Co., Cat. #24510) conjugated to EDC.

The linkers described above contain components that have differentattributes, thus leading to conjugates with differing physico-chemicalproperties. For example, sulfo-NHS esters of alkyl carboxylates are morestable than sulfo-NHS esters of aromatic carboxylates. NHS-estercontaining linkers are less soluble than sulfo-NHS esters. Further, thelinker SMPT contains a sterically hindered disulfide bond, and can formconjugates with increased stability. Disulfide linkages, are in general,less stable than other linkages because the disulfide linkage is cleavedin vitro, resulting in less conjugate available. Sulfo-NHS, inparticular, can enhance the stability of carbodimide couplings.Carbodimide couplings (such as EDC) when used in conjunction withsulfo-NHS, forms esters that are more resistant to hydrolysis than thecarbodimide coupling reaction alone.

In one aspect, the conjugate described herein includes an isolatedNaPi2b-targeted antibody that specifically binds to the extracellularregion of SLC34A2 connected directly or indirectly to one or moreD-carrying polymeric scaffolds independently comprisingpoly(l-hydroxymethylethylene hydroxymethyl-formal) (PHF) having amolecular weight ranging from about 2 kDa to about 40 kDa, wherein eachof the one or more D-carrying polymeric scaffolds independently is ofFormula (Ic):

wherein:

each occurrence of D, independently, is a therapeutic or diagnosticagent; L^(D1) is a carbonyl-containing moiety;

each occurrence of

is independently a first linker that contains a biodegradable bond sothat when the bond is broken, D is released in an active form for itsintended therapeutic effect; and the

between L^(D1) and D denotes direct or indirect attachment of D toL^(D1);

each occurrence of

is independently a second linker not yet connected to the isolatedNaPi2b antibody, in which L^(P2) is a moiety containing a functionalgroup that is yet to form a covalent bond with a functional group of theisolated antibody, and the

between L^(D1) and L^(P2) denotes direct or indirect attachment ofL^(P2) to L^(D1), and each occurrence of the second linker is distinctfrom each occurrence of the first linker;

each occurrence of

is independently a third linker that connects each D-carrying polymericscaffold to the isolated antibody, in which the terminal

attached to L² denotes direct or indirect attachment of L^(P2) to theisolated antibody upon formation of a covalent bond between a functionalgroup of L^(P2) and a functional group of the isolated antibody; andeach occurrence of the third linker is distinct from each occurrence ofthe first linker;

m is an integer from 1 to about 300,

m₁ is an integer from 1 to about 140,

m₂ is an integer from 1 to about 40,

m₃ is an integer from 0 to about 18,

m₄ is an integer from 1 to about 10;

the sum of m, m₁, m₂, m₃, and m₄ ranges from about 15 to about 300; andthe total number of L^(P2) attached to the isolated antibody is 10 orless.

The conjugate may include one or more of the following features.

For example, in Formula (Ic), m₁ is an integer from 1 to about 120(e.g., about 1-90) and/or m₃ is an integer from 1 to about 10 (e.g.,about 1-8).

For example, when the PHF in Formula (Ic) has a molecular weight rangingfrom about 6 kDa to about 20 kDa (i.e., the sum of m, m₁, m₂, m₃, and m₄ranging from about 45 to about 150), m₂ is an integer from 2 to about20, m₃ is an integer from 0 to about 9, m₄ is an integer from 1 to about10, and/or m₁ is an integer from 1 to about 75 (e.g., m₁ being about4-45).

For example, when the PHF in Formula (Ic) has a molecular weight rangingfrom about 8 kDa to about 15 kDa (i.e., the sum of m, m₁, m₂, m₃, and m₄ranging from about 60 to about 110), m₂ is an integer from 2 to about15, m₃ is an integer from 0 to about 7, m₄ is an integer from 1 to about10, and/or m₁ is an integer from 1 to about 55 (e.g., m₁ being about4-30).

For example, when the PHF in Formula (Ic) has a molecular weight rangingfrom about 2 kDa to about 20 kDa (i.e., the sum of m, m₁, m₂, m₃, and m₄ranging from about 15 to about 150), m₂ is an integer from 1 to about20, m₃ is an integer from 0 to about 10 (e.g., m₃ ranging from 0 toabout 9), 1114 is an integer from 1 to about 8, and/or m₁ is an integerfrom 1 to about 70, and the total number of L¹² connected to theisolated antibody ranges from about 2 to about 8 (e.g., about 2, 3, 4,5, 6, 7, or 8).

For example, when the PHF in Formula (Ic) has a molecular weight rangingfrom about 3 kDa to about 15 kDa (i.e., the sum of m, m₁, m₂, m₃, and m₄ranging from about 20 to about 110), m₂ is an integer from 2 to about15, m₃ is an integer from 0 to about 8 (e.g., m₃ ranging from 0 to about7), m₄ is an integer from 1 to about 8, and/or m₁ is an integer from 2to about 50, and the total number of L¹² connected to the isolatedantibody ranges from about 2 to about 8 (e.g., about 2, 3, 4, 5, 6, 7,or 8).

For example, when the PHF in Formula (Ic) has a molecular weight rangingfrom about 5 kDa to about 10 kDa, (i.e., the sum of m, m₁, m₂, m₃ and m₄ranges from about 40 to about 75), m₂ is an integer from about 2 toabout 10 (e.g., m₂ being about 3-10), m₃ is an integer from 0 to about 5(e.g., m₃ ranging from 0 to about 4), m₄ is an integer from 1 to about 8(e.g., m₄ ranging from 1 to about 5), and/or m₁ is an integer from about2 to about 35 (e.g., m₁ being about 5-35), and the total number of L¹²connected to the isolated antibody ranges from about 2 to about 8 (e.g.,about 2, 3, 4, 5, 6, 7, or 8).

For example, when the PHF has a molecular weight ranging from 2 kDa to40 kDa, (e.g., about 6-20 kDa or about 8-15 kDa, about 2-20 kDa, orabout 3-15 kDa, or about 5-10 kDa), the number of drugs per PHF (e.g.,m₂) is an integer from 1 to about 40 (e.g., about 1-20, or about 2-15 orabout 3-10 or about 2-10). This scaffold can be used, for example, forconjugating the isolated antibody having a molecular weight of 40 kDa orgreater (e.g., 60 kDa or greater; 80 kDa or greater; 100 kDa or greater;120 kDa or greater; 140 kDa or greater; 160 kDa or greater, 180 kDa orgreater, or 200 kDa or greater, or about 40-200 kDa, 40-180 kDa, 40-140kDa, 60-200 kDa, 60-180 kDa, 60-140 kDa, 80-200 kDa, 80-180 kDa, 80-140kDa, 100-200 kDa, 100-180 kDa, 100-140 kDa or 140-150 kDa). In thisembodiment, the ratio of the isolated antibody to PHF is between about1:1 and about 1:10, between about 1:1 and about 1:9, between about 1:1and about 1:8, between about 1:1 and about 1:7, between about 1:1 andabout 1:6, between about 1:1 and about 1:5, between about 1:1 and about1:4, between about 1:1 and about 1:3, between about 1:1 and about 1:2,between about 1:2 and about 1:4, between about 1:2 and about 1:3,between about 1:3 and about 1:4, or between about 1:3 and about 1:5.

For example, when the PHF has a molecular weight ranging from 2 kDa to40 kDa, (e.g., about 6-20 kDa or about 8-15 kDa, about 2-20 kDa, orabout 3-15 kDa, or about 5-10 kDa), the number of drugs per PHF (e.g.,m₂) is an integer from 1 to about 40 (e.g., about 1-20, or about 2-15 orabout 3-10 or about 2-10). This scaffold can be used, for example, forconjugating the isolated antibody having a molecular weight of 140 kDato 180 kDa or of 140 kDa to 150 kDa. In this embodiment, the ratio ofthe isolated antibody to PHF is between about 1:1 and about 1:10,between about 1:1 and about 1:9, between about 1:1 and about 1:8,between about 1:1 and about 1:7, between about 1:1 and about 1:6,between about 1:1 and about 1:5, between about 1:1 and about 1:4,between about 1:1 and about 1:3, between about 1:1 and about 1:2,between about 1:2 and about 1:4, between about 1:2 and about 1:3,between about 1:3 and about 1:4, or between about 1:3 and about 1:5.

The NaPi-2b antibody in this molecular weight range, include but are notlimited to, for example, full length antibodies, such as, IgG, IgM.

For example, when the PHF has a molecular weight ranging from 2 kDa to40 kDa, the number of drugs per PHF (e.g., m₂) is an integer from 1 toabout 40 (e.g., about 1-20 or about 2-15 or about 3-10 or about 2-10).This scaffold can be used, for example, for conjugating the isolatedantibody having a molecular weight of 60 kDa to 120 kDa. In thisembodiment, the ratio of the isolated antibody to PHF is between about1:1 and about 1:10, between about 1:1 and about 1:9, between about 1:1and about 1:8, between about 1:1 and about 1:7, between about 1:1 andabout 1:6, between about 1:1 and about 1:5, between about 1:1 and about1:4, between about 1:1 and about 1:3, between about 1:1 and about 1:2,between about 1:2 and about 1:4, between about 1:2 and about 1:3,between about 1:3 and about 1:4, or between about 1:3 and about 1:5.

In certain embodiment, D is a therapeutic agent. In certain embodiments,the therapeutic agent is a small molecule having a molecular weight≤about 5 kDa, ≤about 4 kDa, ≤about 3 kDa, ≤about 1.5 kDa, or ≤about 1kDa.

In certain embodiments, the therapeutic agent has an IC₅₀ of about lessthan 1 nM.

In another embodiment, the therapeutic agent has an IC₅₀ of aboutgreater than 1 nM, for example, the therapeutic agent has an IC₅₀ ofabout 1 to 50 nM.

Some therapeutic agents having an IC₅₀ of greater than about 1 nM (e.g.,“less potent drugs”) are unsuitable for conjugation with an antibodyusing art-recognized conjugation techniques. Without wishing to be boundby theory, such therapeutic agents have a potency that is insufficientfor use in targeted antibody-drug conjugates using conventionaltechniques as sufficient copies of the drug (i.e., more than 8) cannotbe conjugated using art-recognized techniques without resulting indiminished pharmacokinetic and physiochemical properties of theconjugate. However sufficiently high loadings of these less potent drugscan be achieved using the conjugation strategies described hereinthereby resulting in high loadings of the therapeutic agent whilemaintaining the desirable pharmacokinetic and physiochemical properties.Thus, the disclosure also relates to an antibody-polymer-drug conjugatethat includes the isolated antibody, PHF and at least eight therapeuticagent moieties, where D is auristatin, dolastatin (e.g., dolastatin 10or dolastatin 15), monomethylauristatin E (MMAE), monomethylauristatin F(MMAF), auristatin F, AF HPA, monomethyl AF HPA, phenylenediamine (AFP).

For example, the duocarmycin or analogs thereof is duocarmycin A,duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2,duocarmycin D, duocarmycin SA, CC-1065, adozelesin, bizelesin, orcarzelesin.

Other examples of D include those described in, for example, U.S. Pat.No. 8,815,226; and US Application Publication No. 2015-0104407; thecontents of each of which are incorporated herein in their entireties.

In some embodiments, the number of D-carrying polymeric scaffolds thatcan be conjugated to an antibody is limited by the number of freecysteine residues. In some embodiments, free cysteine residues areintroduced into the antibody amino acid sequence by the methodsdescribed herein. Exemplary conjugates disclosed herein can includeantibodies that have 1, 2, 3, or 4 engineered cysteine amino acids(Lyon, R. et al (2012) Methods in Enzym. 502:123-138). In someembodiments, one or more free cysteine residues are already present inan antibody, without the use of engineering, in which case the existingfree cysteine residues may be used to conjugate the antibody to aD-carrying polymeric scaffold. In some embodiments, an antibody isexposed to reducing conditions prior to conjugation of the antibody inorder to generate one or more free cysteine residues.

In certain embodiments, the functional group of L^(P2) that is yet toform a covalent bond with a functional group of the isolated antibody(such as a functional group or a reactive moiety on an amino acidresidue of the antibody, for example, a functional group on a cysteineresidue or a lysine residue of the antibody), is selected from —SR^(p),—S—S-LG,

and halo, in which LG is a leaving group, R^(p) is H or a sulfurprotecting group, and one of X_(a) and X_(b) is H and the other is awater-soluble maleimido blocking moiety, or X_(a) and X_(b), togetherwith the carbon atoms to which they are attached for a carbon-carbondouble bond. For example, the functional group of L^(P2) that is yet toform a covalent bond is a functional group that is not reacted with afunctional group of the isolated antibody, e.g.,

as the functional group of L^(P2), in which one of X_(a) and X_(b) is Hand the other is a water-soluble maleimido blocking moiety, or X_(a) andX_(b).

In certain embodiments, in the conjugate described herein, theD-carrying polymeric scaffold of Formula (Ic) is of Formula (Ie):

wherein,

the PHF has a molecular weight ranging from about 2 kDa to about 40 kDa;

each occurrence of D independently is a therapeutic agent having amolecular weight of ≤5 kDa, and the

between D and the carbonyl group denotes direct or indirect attachmentof D to the carbonyl group,

X is CH₂, O, or NH;

-   -   one of X_(a) and X_(b) is H and the other is a water-soluble        maleimido blocking moiety, or X_(a) and X_(b), together with the        carbon atoms to which they are attached for a carbon-carbon        double bond, m₁ is an integer from 1 to about 140,    -   m₇ is an integer from 1 to about 40, and the sum of m₁ and m₇ is        about 2 to about 180    -   m is an integer from 1 to about 300,    -   m_(3a) is an integer from 0 to about 17,    -   m_(3b) is an integer from 1 to about 8, and the sum of m_(3a)        and m_(3b) is between 1 and 18, and    -   the sum of m, m₁, m₇, m₃a, and m_(3b) ranges from about 15 to        about 300.

In certain embodiments, in the conjugate described herein, theD-carrying polymeric scaffold of Formula (Ie) is of Formula (Id):

wherein:

one of X_(a) and X_(b) is H and the other is a water-soluble maleimidoblocking moiety, or X_(a) and X_(b), together with the carbon atoms towhich they are attached for a carbon-carbon double bond;

m_(3a) is an integer from 0 to about 17,

m_(3b) is an integer from 1 to about 8, and the sum of m_(3a) and m_(3b)is between 1 and 18, and

the sum of m, m₁, m₂, m₃a, and m_(3b) ranges from about 15 to about 300.

For example, the ratio between m₂ and m_(3b) is greater than 1:1 andless than or equal to 10:1.

For example, the ratio between m₂ and m_(3b) is about 9:1, 8:1, 7:1,6:1, 5:1, 4:1, 3:1, or 2:1.

For example, the ratio between m₂ and m_(3b) is between 2:1 and 8:1.

For example, the ratio between m₂ and m_(3b) is about 8:1, 7:1, 6:1,5:1, 4:1, 3:1, or 2:1.

For example, the ratio between m₂ and m_(3b) is between 2:1 and 4:1.

For example, the ratio between m₂ and m_(3b) is about 4:1, 3:1, or 2:1.

For example, the ratio between m₂ and m_(3b) is about 3:1 and 5:1.

For example, the ratio between m₂ and m_(3b) is about 3:1, 4:1 or 5:1.

For example, when the PHF in Formula (Id) has a molecular weight rangingfrom about 2 kDa to about 20 kDa, the sum of m, m₁, m₂, m_(3a) andm_(3b) ranges from about 15 to about 150, m₁ is an integer from 1 toabout 70, m₂ is an integer from 1 to about 20, m_(3a) is an integer from0 to about 9, m_(3b) is an integer from 1 to about 8 and the ratiobetween the PHF and the isolated NaPi2b-targeted antibody is an integerfrom 2 to about 8.

For example, when the PHF in Formula (Id) has a molecular weight rangingfrom about 3 kDa to about 15 kDa, the sum of m, m₁, m₂, m_(3a) andm_(3b) ranges from about 20 to about 110, m₁ is an integer from 2 toabout 50, m₂ is an integer from 2 to about 15, m_(3a) is an integer from0 to about 7, m_(3b) is an integer from 1 to about 8 and the ratiobetween the PHF and the isolated NaPi2b-targeted antibody is an integerfrom 2 to about 8 (e.g., an integer from 2 to about 6 or an integer from2 to about 4).

For example, when the PHF in Formula (Id) has a molecular weight rangingfrom about 5 kDa to about 10 kDa, the sum of m, m₁, m₂, m_(3a) andm_(3b) ranges from about 40 to about 75, m₁ is an integer from about 2to about 35, m₂ is an integer from about 2 to about 10, m_(3a) is aninteger from 0 to about 4, m_(3b) is an integer from 1 to about 5 andthe ratio between the PHF and the isolated NaPi2b-targeted antibody isan integer from 2 to about 8 (e.g., an integer from 2 to about 6 or aninteger from 2 to about 4).

For example, the water-soluble maleimido blocking moieties are moietiesthat can be covalently attached to one of the two olefin carbon atomsupon reaction of the maleimido group with a thiol-containing compound ofFormula (II):

R₉₀—(CH₂)_(d)—SH   (II)

wherein:

R₉₀ is NHR₉₁, OH, COOR₉₃, CH(NHR₉₁)COOR₉₃ or a substituted phenyl group;

R₉₃ is hydrogen or C₁₋₄ alkyl;

R₉₁ is hydrogen, CH₃ or CH₃CO and

d is an integer from 1 to 3.

For example, the water-soluble maleimido blocking compound of Formula(II) can be cysteine, N-acetyl cysteine, cysteine methyl ester, N-methylcysteine, 2-mercaptoethanol, 3-mercaptopropanoic acid, 2-mercaptoaceticacid, mercaptomethanol (i.e., HOCH₂SH), benzyl thiol in which phenyl issubstituted with one or more hydrophilic substituents, or3-aminopropane-1-thiol. The one or more hydrophilic substituents onphenyl comprise OH, SH, methoxy, ethoxy, COOH, CHO, COC₁₋₄ alkyl, NH₂,F, cyano, SO₃H, PO₃H, and the like.

For example, the water-soluble maleimido blocking group is—S—(CH₂)_(d)—R₉₀, in which, R₉₀ is OH, COOH, or CH(NHR₉₁)COOR₉₃;

R₉₃ is hydrogen or CH₃;

R₉₁ is hydrogen or CH₃CO; and

d is 1 or 2.

For example, the water-soluble maleimido blocking group is—S—CH₂—CH(NH₂)COOH.

For example, when the PHF has a molecular weight ranging from 2 kDa to40 kDa, (e.g., about 2-20 kDa, or about 3-15 kDa, or about 5-10 kDa),the number of drugs per PHF (e.g., m₂) is an integer from 1 to about 40(e.g., about 1-20 or about 2-15 or about 3-10 or about 2-10). Thisscaffold can be used, for example, for conjugating an isolated antibodyhaving a molecular weight of 40 kDa or greater (e.g., 60 kDa or greater;80 kDa or greater; or 100 kDa or greater; 120 kDa or greater; 140 kDa orgreater; 160 kDa or greater, 180 kDa or greater, or 200 kDa or greater,or about 40-200 kDa, 40-180 kDa, 40-140 kDa, 60-200 kDa, 60-180 kDa,60-140 kDa, 80-200 kDa, 80-180 kDa, 80-140 kDa, 100-200 kDa, 100-180kDa, 100-140 kDa or 140-150 kDa). In this embodiment, the ratio of theisolated antibody to PHF is between about 1:1 and about 1:10, betweenabout 1:1 and about 1:9, between about 1:1 and about 1:8, between about1:1 and about 1:7, between about 1:1 and about 1:6, between about 1:1and about 1:5, between about 1:1 and about 1:4, between about 1:1 andabout 1:3, between about 1:1 and about 1:2, between about 1:2 and about1:8, between about 1:2 and about 1:6, between about 1:2 and about 1:5,between about 1:2 and about 1:4, between about 1:2 and about 1:3,between about 1:3 and about 1:4, or between about 1:3 and about 1:5.

For example, when the PHF has a molecular weight ranging from 2 kDa to40 kDa, (e.g., about 2-20 kDa, or about 3-15 kDa, or about 5-10 kDa),the number of drugs per PHF (e.g., m₂) is an integer from 1 to about 40(e.g., about 1-20 or about 2-15 or about 3-10 or about 2-10). Thisscaffold can be used, for example, for conjugating an isolated antibodyhaving a molecular weight of 140 kDa to 180 kDa or of 140 kDa to 150kDa. In this embodiment the ratio of the isolated antibody to PHF isbetween about 1:1 and about 1:10, between about 1:1 and about 1:9,between about 1:1 and about 1:8, between about 1:1 and about 1:7,between about 1:1 and about 1:6, between about 1:1 and about 1:5,between about 1:1 and about 1:4, between about 1:1 and about 1:3,between about 1:1 and about 1:2, between about 1:2 and about 1:8,between about 1:2 and about 1:6, between about 1:2 and about 1:5,between about 1:2 and about 1:4, between about 1:2 and about 1:3,between about 1:3 and about 1:4, or between about 1:3 and about 1:5.

The isolated antibodies in this molecular weight range, include but arenot limited to, for example, full length antibodies, such as, IgG, IgM.

For example, when the PHF has a molecular weight ranging from 2 kDa to40 kDa (e.g., about 2-20 kDa, or about 3-15 kDa, or about 5-10 kDa), thenumber of drugs per PHF (e.g., m₂) is an integer from 1 to about 40,(e.g., about 1-20 or about 2-15 or about 3-10 or 2-10). This scaffoldcan be used, for example, for conjugating an isolated antibody having amolecular weight of 60 kDa to 120 kDa. In this embodiment the ratio ofthe isolated antibody to PHF is between about 1:1 and about 1:10,between about 1:1 and about 1:9, between about 1:1 and about 1:8,between about 1:1 and about 1:7, between about 1:1 and about 1:6,between about 1:1 and about 1:5, between about 1:1 and about 1:4,between about 1:1 and about 1:3, between about 1:1 and about 1:2,between about 1:2 and about 1:8, between about 1:2 and about 1:6,between about 1:2 and about 1:5, between about 1:2 and about 1:4,between about 1:2 and about 1:3, between about 1:3 and about 1:4, orbetween about 1:3 and about 1:5.

For example, when the PHF has a molecular weight ranging from 2 kDa to40 kDa, (e.g., about 2-20 kDa, or about 3-15 kDa, or about 5-10 kDa),the number of drugs per PHF (e.g., m₂) is an integer from 1 to about 40(e.g., about 1-20 or about 2-15 or about 3-10 or 2-10). This scaffoldcan be used, for example, for conjugating the isolated antibody having amolecular weight of 40 kDa to 80 kDa. In this embodiment the ratio ofthe isolated antibody to PHF is between about 1:1 and about 1:10,between about 1:1 and about 1:9, between about 1:1 and about 1:8,between about 1:1 and about 1:7, between about 1:1 and about 1:6,between about 1:1 and about 1:5, between about 1:1 and about 1:4,between about 1:1 and about 1:3, between about 1:1 and about 1:2,between about 1:2 and about 1:8, between about 1:2 and about 1:6,between about 1:2 and about 1:5, between about 1:2 and about 1:4,between about 1:2 and about 1:3, between about 1:3 and about 1:4, orbetween about 1:3 and about 1:5.

In certain embodiments, in the conjugate described herein, theD-carrying polymeric scaffold of Formula (Ie) is of Formula (If),wherein the polymer is PHF that has a molecular weight ranging fromabout 2 kDa to about 40 kDa:

wherein:

m is an integer from 1 to about 300,

m₁ is an integer from 1 to about 140,

m₂ is an integer from 1 to about 40,

m_(3a) is an integer from 0 to about 17,

m_(3b) is an integer from 1 to about 8;

the sum of m_(3a) and m_(3b) ranges from 1 and about 18;

the sum of m, m₁, m₂, m_(3a), and m_(3b) ranges from about 15 to about300;

the terminal

denotes the attachment of one or more polymeric scaffolds to theisolated antibody that specifically binds to the extracellular region ofSLC34A2 (i) a variable heavy chain complementarity determining region 1(CDRH1) comprising the amino acid sequence GYTFTGYNIH (SEQ ID NO: 5); avariable heavy chain complementarity determining region 2 (CDRH2)comprising the amino acid sequence AIYPGNGDTSYKQKFRG (SEQ ID NO: 6); avariable heavy chain complementarity determining region 3 (CDRH3)comprising the amino acid sequence GETARATFAY (SEQ ID NO: 7); a variablelight chain complementarity determining region 1 (CDRL1) comprising theamino acid sequence SASQDIGNFLN (SEQ ID NO: 8); a variable light chaincomplementarity determining region 2 (CDRL2) comprising the amino acidsequence YTSSLYS (SEQ ID NO: 9); and a variable light chaincomplementarity determining region 3 (CDRL3) comprising the amino acidsequence QQYSKLPLT (SEQ ID NO: 10) or (ii) a CDRH1 comprising the aminoacid sequence GFSFSDFAMS (SEQ ID NO: 18); a CDRH2 comprising the aminoacid sequence ATIGRVAFHTYYPDSMKG (SEQ ID NO: 19); a CDRH3 comprising theamino acid sequence ARHRGFDVGHFDF (SEQ ID NO: 20); a CDRL1 comprisingthe amino acid sequence RSSETLVHSSGNTYLE (SEQ ID NO: 21); a CDRL2comprising the amino acid sequence RVSNRFS (SEQ ID NO: 22); and a CDRL3comprising the amino acid sequence FQGSFNPLT (SEQ ID NO: 23); and theratio between the PHF and the antibody is 10 or less.

In some embodiments, the isolated NaPi2b-targeted antibody specificallybinds to SLC34A2 and includes (i) a CDRH1 comprising the amino acidsequence GYTFTGYNIH (SEQ ID NO: 5); a CDRH2 comprising the amino acidsequence AIYPGNGDTSYKQKFRG (SEQ ID NO: 6); a CDRH3 comprising the aminoacid sequence GETARATFAY (SEQ ID NO: 7); a CDRL1 comprising the aminoacid sequence SASQDIGNFLN (SEQ ID NO: 8); a CDRL2 comprising the aminoacid sequence YTSSLYS (SEQ ID NO: 9); and a CDRL3 comprising the aminoacid sequence QQYSKLPLT (SEQ ID NO: 10).

In some embodiments, the isolated NaPi2b-targeted antibody specificallybinds to SLC34A2 and includes a CDRH1 comprising the amino acid sequenceGFSFSDFAMS (SEQ ID NO: 18); a CDRH2 comprising the amino acid sequenceATIGRVAFHTYYPDSMKG (SEQ ID NO: 19); a CDRH3 comprising the amino acidsequence ARHRGFDVGHFDF (SEQ ID NO: 20); a CDRL1 comprising the aminoacid sequence RSSETLVHSSGNTYLE (SEQ ID NO: 21); a CDRL2 comprising theamino acid sequence RVSNRFS (SEQ ID NO: 22); and a CDRL3 comprising theamino acid sequence FQGSFNPLT (SEQ ID NO: 23).

The scaffold of Formula (If) can include one or more of the followingfeatures:

When the PHF in Formula (If) has a molecular weight ranging from about 2kDa to about 20 kDa, the sum of m, m₁, m₂, m_(3a) and m_(3b) ranges fromabout 15 to about 150, m₁ is an integer from 1 to about 70, m₂ is aninteger from 1 to about 20, m_(3a) is an integer from 0 to about 9,m_(3b) is an integer from 1 to about 8, the sum of m_(3a) and m_(3b)ranges from 1 and about 10, and the ratio between the PHF and antibodyis an integer from 2 to about 8.

When the PHF in Formula (If) has a molecular weight ranging from about 3kDa to about 15 kDa, the sum of m, m₁, m₂, m_(3a) and m_(3b) ranges fromabout 20 to about 110, m₁ is an integer from 2 to about 50, m₂ is aninteger from 2 to about 15, m_(3a) is an integer from 0 to about 7,m_(3b) is an integer from 1 to about 8, the sum of m_(3a) and m_(3b)ranges from 1 and about 8; and the ratio between the PHF and antibody isan integer from 2 to about 8 (e.g., from about 2 to about 6 or fromabout 2 to about 4).

When the PHF in Formula (If) has a molecular weight ranging from about 5kDa to about 10 kDa, the sum of m, m₁, m₂, m_(3a) and m_(3b) ranges fromabout 40 to about 75, m₁ is an integer from about 2 to about 35, m₂ isan integer from about 2 to about 10, m_(3a) is an integer from 0 toabout 4, m_(3b) is an integer from 1 to about 5, the sum of m_(3a) andm_(3b) ranges from 1 and about 5; and the ratio between the PHF andantibody is an integer from 2 to about 8 (e.g., from about 2 to about 6or from about 2 to about 4).

In certain embodiments, the ratio between auristatin F hydroxypropylamide (“AF HPA”) and the antibody can be about 30:1, 29:1, 28:1, 27:1,26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1,14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1 or 6:1.

In certain embodiments, the ratio between AF HPA and the antibody can beabout 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1,14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1 or 6:1.

In other embodiments, the ratio between AF HPA and the antibody can beabout 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1,9:1, 8:1, 7:1 or 6:1.

In some embodiments, the ratio between AF HPA and the antibody can beabout 16:1, 15:1, 14:1, 13:1, 12:1, 11:1 or 10:1.

In some embodiments, the ratio between AF and the antibody can be about15:1, 14:1, 13:1, 12:1 or 11:1.

In some embodiments, the ratio between AF HPA and the antibody can beabout 15:1, 14:1, 13:1 or 12:1.

In certain embodiments, the ratio between PHF and the antibody can beabout 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1 or 1:1.

In certain embodiments, the ratio between PHF and the antibody can beabout 8:1, 7:1, 6:1, 5:1, 4:1, 3:1 or 2:1.

In other embodiments, the ratio between PHF and the antibody can beabout 6:1, 5:1, 4:1, 3:1, 2:1 or 1:1.

In other embodiments, the ratio between PHF and the antibody can beabout 6:1, 5:1, 4:1, 3:1 or 2:1.

In other embodiments, the ratio between PHF and the antibody can beabout 6:1, 5:1, 4:1 or 3:1.

In some embodiments, the ratio between PHF and the antibody can be about5:1, 4:1 or 3:1.

In some embodiments, the ratio between PHF and the antibody can be about4:1, 3:1 or 2:1.

Other embodiments of antibody-polymer drug conjugates are thosedescribed in, for example, U.S. Pat. No. 8,815,226; and US ApplicationPublication No. 2015-0104407; the contents of each of which areincorporated herein in their entireties.

This disclosure also relates to a drug derivative so modified that itcan be directly conjugated to an antibody absent a polymeric carrier,and the drug-antibody conjugates thereof.

In some embodiments, the NaPi2b-targeted antibody drug conjugatesinclude an antibody conjugated, i.e., covalently attached, to the drugmoiety. In some embodiments, the antibody is covalently attached to thedrug moiety through a linker, e.g., a non-polymeric linker.

The “D” (e.g., a drug moiety) of the NaPi2b-targeted antibody-drugconjugates (ADC) may include any compound, moiety or group that has acytotoxic or cytostatic effect as defined herein. In certainembodiments, a NaPi2b-targeted-antibody-drug conjugate (ADC) comprises aNaPi2b-targeted antibody (Ab) which targets a tumor cell, D (e.g., adrug moiety), and a linker moiety (L) that attaches Ab to D. In someembodiments, the antibody is attached to the linker moiety (L) throughone or more amino acid residues, such as lysine and/or cysteine.

In certain embodiments the ADC has Formula (Ig):

Ab-(L-D)_(p)   (Ig),

where p is 1 to about 20.

In some embodiments, the number of drug moieties that can be conjugatedto an antibody is limited by the number of free cysteine residues. Insome embodiments, free cysteine residues are introduced into theantibody amino acid sequence by the methods described herein. ExemplaryADC of Formula Ig include, but are not limited to, antibodies that have1, 2, 3, or 4 engineered cysteine amino acids (Lyon, R. et al (2012)Methods in Enzym. 502:123-138). In some embodiments, one or more freecysteine residues are already present in an antibody, without the use ofengineering, in which case the existing free cysteine residues may beused to conjugate the antibody to a drug. In some embodiments, anantibody is exposed to reducing conditions prior to conjugation of theantibody in order to generate one or more free cysteine residues.

In some embodiments the “Linker” (L) is a bifunctional ormultifunctional moiety that can be used to link one or more D (e.g.,drug moieties) to an antibody (Ab) to form an antibody-drug conjugate(ADC) of Formula Ig. In some embodiments, antibody-drug conjugates (ADC)can be prepared using a Linker having reactive functionalities forcovalently attaching to the drug and to the antibody. For example, insome embodiments, a cysteine thiol of an antibody (Ab) can form a bondwith a reactive functional group of a linker or a drug-linkerintermediate to make an ADC.

In one aspect, a linker has a functionality that is capable of reactingwith a free cysteine present on an antibody to form a covalent bond.Nonlimiting exemplary such reactive functionalities include maleimide,haloacetamides, α-haloacetyl, activated esters such as succinimideesters, 4-nitrophenyl esters, pentafluorophenyl esters,tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonylchlorides, isocyanates, and isothiocyanates. See, e.g., the conjugationmethod at page 766 of Klussman, et al (2004), Bioconjugate Chemistry15(4):765-773, and the Examples herein.

In some embodiments, a linker has a functionality that is capable ofreacting with an electrophilic group present on an antibody. Exemplarysuch electrophilic groups include, but are not limited to, aldehyde andketone carbonyl groups. In some embodiments, a heteroatom of thereactive functionality of the linker can react with an electrophilicgroup on an antibody and form a covalent bond to an antibody unit.Nonlimiting exemplary such reactive functionalities include, but are notlimited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone,hydrazine carboxylate, and arylhydrazide.

A linker may comprise one or more linker components. Exemplary linkercomponents include 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”),valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine(“ala-phe”), p-aminobenzyloxycarbonyl (a “PAB”), N-Succinimidyl4-(2-pyridylthio) pentanoate (“SPP”), and4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“MCC”). Various linkercomponents are known in the art, some of which are described below.

A linker may be a “cleavable linker,” facilitating release of a drug.Nonlimiting exemplary cleavable linkers include acid-labile linkers(e.g., comprising hydrazone), protease-sensitive (e.g.,peptidase-sensitive) linkers, photolabile linkers, ordisulfide-containing linkers (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020).

In certain embodiments, a linker has the following Formula (IIg):

-A_(a)-W_(w)—Y_(y)   (IIg)

wherein:

A is a “stretcher unit”, and a is an integer from 0 to 1;

W is an “amino acid unit”, and w is an integer from 0 to 12;

Y is a “spacer unit”, and y is an integer 0, 1, or 2. An ADC comprisingthe linker of Formula (IIg) has the Formula I(A): Ab-(Aa-Ww-Yy-D)p,wherein Ab, D, and p are defined as above for Formula (Ig). Exemplaryembodiments of such linkers are described in U.S. Pat. No. 7,498,298,which is incorporated herein by reference in its entirety.

In some embodiments, a linker component comprises a “stretcher unit” (A)that links an antibody to another linker component or to a drug moiety.Nonlimiting exemplary stretcher units are shown below (wherein the wavyline indicates sites of covalent attachment to an antibody, drug, oradditional linker components):

In some embodiments, a linker component comprises an “amino acid unit”(W). In some such embodiments, the amino acid unit allows for cleavageof the linker by a protease, thereby facilitating release of the drugfrom the immunoconjugate upon exposure to intracellular proteases, suchas lysosomal enzymes (Doronina et al. (2003) Nat. Biotechnol.21:778-784). Exemplary amino acid units include, but are not limited to,dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplarydipeptides include, but are not limited to, valine-citrulline (vc orval-cit), alanine-phenylalanine (af or ala-phe); phenylalanine-lysine(fk or phe-lys); phenylalanine-homolysine (phe-homolys); andN-methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides include,but are not limited to, glycine-valine-citrulline (gly-val-cit) andglycine-glycine-glycine (gly-gly-gly). An amino acid unit may compriseamino acid residues that occur naturally and/or minor amino acids and/ornon-naturally occurring amino acid analogs, such as citrulline. Aminoacid units can be designed and optimized for enzymatic cleavage by aparticular enzyme, for example, a tumor-associated protease, cathepsinB, C and D, or a plasmin protease.

Typically, peptide-type linkers can be prepared by forming a peptidebond between two or more amino acids and/or peptide fragments. Suchpeptide bonds can be prepared, for example, according to a liquid phasesynthesis method (e.g., E. Schrider and K. Lubke (1965) “The Peptides”,volume 1, pp 76-136, Academic Press).

In some embodiments, a linker component comprises a “spacer” unit thatlinks the antibody to a drug moiety, either directly or through astretcher unit and/or an amino acid unit. A spacer unit may be“self-immolative” or a “non-self-immolative.” A “non-self-immolative”spacer unit is one in which part or all of the spacer unit remains boundto the drug moiety upon cleavage of the ADC. Examples ofnon-self-immolative spacer units include, but are not limited to, aglycine spacer unit and a glycine-glycine spacer unit. In someembodiments, enzymatic cleavage of an ADC containing a glycine-glycinespacer unit by a tumor-cell associated protease results in release of aglycine-glycine-drug moiety from the remainder of the ADC. In some suchembodiments, the glycine-glycine-drug moiety is subjected to ahydrolysis step in the tumor cell, thus cleaving the glycine-glycinespacer unit from the drug moiety.

A “self-immolative” spacer unit allows for release of the drug moiety.In certain embodiments, a spacer unit of a linker comprises ap-aminobenzyl unit. In some such embodiments, a p-aminobenzyl alcohol isattached to an amino acid unit via an amide bond, and a carbamate,methylcarbamate, or carbonate is made between the benzyl alcohol and thedrug (Hamann et al. (2005) Expert Opin. Ther. Patents (2005)15:1087-1103). In some embodiments, the spacer unit comprisesp-aminobenzyloxycarbonyl (PAB). In some embodiments, an ADC comprising aself-immolative linker has the structure:

wherein:

Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), halogen, nitro, or cyano;

n₆ is an integer from 0 to 4;

X_(a) may be one or more additional spacer units or may be absent; and

p in an integer from 1 to about 20.

In some embodiments, p in an integer from 1 to 10, 1 to 7, 1 to 5, or 1to 4. Nonlimiting exemplary X_(a) spacer units include:

wherein R₁₀₁ and R₁₀₂ are independently selected from H and C₁-C₆ alkyl.In some embodiments, R₁₀₁ and R₁₀₂ are each —CH₃.

Other examples of self-immolative spacers include, but are not limitedto, aromatic compounds that are electronically similar to the PAB group,such as 2-aminoimidazol-5-methanol derivatives (U.S. Pat. No. 7,375,078;Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and ortho-orpara-aminobenzylacetals. In some embodiments, spacers can be used thatundergo cyclization upon amide bond hydrolysis, such as substituted andunsubstituted 4-aminobutyric acid amides (Rodrigues et al (1995)Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] andbicyclo[2.2.2] ring systems (Storm et al (1972) J. Amer. Chem. Soc.94:5815) and 2-aminophenylpropionic acid amides (Amsberry, et al (1990)J. Org. Chem. 55:5867). Linkage of a drug to the α-carbon of a glycineresidue is another example of a self-immolative spacer that may beuseful in ADC (Kingsbury et al (1984) J. Med. Chem. 27:1447).

In some embodiments, linker L may be a dendritic type linker forcovalent attachment of more than one drug moiety to an antibody througha branching, multifunctional linker moiety (Sun et al (2002) Bioorganic& Medicinal Chemistry Letters 12:2213-2215; Sun et al (2003) Bioorganic& Medicinal Chemistry 11:1761-1768). Dendritic linkers can increase themolar ratio of drug to antibody, i.e., loading, which is related to thepotency of the ADC. Thus, where an antibody bears only one reactivecysteine thiol group, a multitude of drug moieties may be attachedthrough a dendritic linker.

Nonlimiting exemplary linkers are shown below for ADCs of Formula (Ig):

wherein R₁₀₁ and R₁₀₂ are independently selected from H and C1-C₆ alkyl;

n₅ is an integer from 0 to 12.

In some embodiments, n is an integer 2 to 10. In some embodiments, n isan integer from 4 to 8.

In some embodiments, R₁₀₁ and R₁₀₂ are each —CH₃.

Further nonlimiting exemplary ADCs include the structures:

wherein Xa is:

Y is:

each R₁₀₃ is independently H or C₁-C₆ alkyl; and n7 is an integer from 1to 12.

In some embodiments, a linker is substituted with groups that modulatesolubility and/or reactivity. As a nonlimiting example, a chargedsubstituent such as sulfonate (—SO₃ ⁻) or ammonium may increase watersolubility of the linker reagent and facilitate the coupling reaction ofthe linker reagent with the antibody and/or the drug moiety, orfacilitate the coupling reaction of Ab-L (antibody-linker intermediate)with D, or D-L (drug-linker intermediate) with Ab, depending on thesynthetic route employed to prepare the ADC. In some embodiments, aportion of the linker is coupled to the antibody and a portion of thelinker is coupled to the drug, and then the Ab-(linker portion)^(a) iscoupled to drug-(linker portion)^(b) to form the ADC of Formula Ig.

The compounds disclosed herein expressly contemplate, but are notlimited to, ADC prepared with the following linker reagents:bis-maleimido-trioxyethylene glycol (BMPEO),N-(β-maleimidopropyloxy)-N-hydroxy succinimide ester (BMPS),N-(ε-maleimidocaproyloxy) succinimide ester (EMCS),N-[γ-maleimidobutyryloxy]succinimide ester (GMBS),1,6-hexane-bis-vinylsulfone (HBVS), succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),4-(4-N-Maleimidophenyl)butyric acid hydrazide (MPBH), succinimidyl3-(bromoacetamido)propionate (SBAP), succinimidyl iodoacetate (SIA),succinimidyl (4-iodoacetyl)aminobenzoate (SIAB),N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), succinimidyl4-(p-maleimidophenyl)butyrate (SMPB), succinimidyl6-[(β-maleimidopropionamido)hexanoate] (SMPH), iminothiolane (IT),sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC,and sulfo-SMPB, and succinimidyl-(4-vinyl sulfone)benzoate (SVSB), andincluding bis-maleimide reagents: dithiobismaleimidoethane (DTME),1,4-Bi smaleimidobutane (BMB), 1,4 Bismaleimidyl-2,3-dihydroxybutane(BMDB), bismaleimidohexane (BMH), bismaleimidoethane (BMOE), BM(PEG)₂(shown below), and BM(PEG)₃ (shown below); bifunctional derivatives ofimidoesters (such as dimethyl adipimidate HCl), active esters (such asdisuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azidocompounds (such as bis-(p-azidobenzoyl) hexanediamine), bis-diazoniumderivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),diisocyanates (such as toluene 2,6-diisocyanate), and bis-activefluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). In someembodiments, bis-maleimide reagents allow the attachment of the thiolgroup of a cysteine in the antibody to a thiol-containing drug moiety,linker, or linker-drug intermediate. Other functional groups that arereactive with thiol groups include, but are not limited to,iodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyldisulfide, isocyanate, and isothiocyanate.

Certain useful linker reagents can be obtained from various commercialsources, such as Pierce Biotechnology, Inc. (Rockford, Ill.), MolecularBiosciences Inc.(Boulder, Colo.), or synthesized in accordance withprocedures described in the art; for example, in Toki et al (2002) J.Org. Chem. 67:1866-1872; Dubowchik, et al. (1997) Tetrahedron Letters,38:5257-60; Walker, M. A. (1995) J. Org. Chem. 60:5352-5355; Frisch etal (1996) Bioconjugate Chem. 7:180-186; U.S. Pat. No. 6,214,345; WO02/088172; US 2003130189; US2003096743; WO 03/026577; WO 03/043583; andWO 04/032828.

Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See, e.g., WO 94/11026.

Methods of Making Polymer NaPi2b-Targeted Antibody Conjugates:

In certain embodiments, the conjugates are formed in several steps.These steps include (1) modifying a polymer so that it contains afunctional group that can react with a functional group of the drug orits derivative; (2) reacting the modified polymer with the drug or itsderivative so that the drug is linked to the polymer; (3) modifying thepolymer-drug conjugate so that the polymer contains a functional groupthat can react with a functional group of the isolated NaPi2b-targetedantibody or its derivative; and (4) reacting the modified polymer-drugconjugate with the NaPi2b-targeted antibody to form the conjugatedisclosed herein. Step (3) may be omitted if the modified polymerproduced by step (1) contains a functional group that can react with afunctional group of the antibody.

In another embodiment the conjugates are formed in several steps: (1)modifying a polymer so that it contains a functional group that canreact with a functional group of a first drug or its derivative; (2)reacting the modified polymer with the first drug or its derivative sothat the first drug is linked to the polymer; (3) modifying thepolymer-drug conjugate so that it contains a different functional groupthat can react with a functional group of a second drug or itsderivative (4) reacting the modified polymer-drug conjugate with thesecond drug or its derivative so that the second drug is linked to thepolymer-drug conjugate; (5) modifying the polymer-drug conjugatecontaining 2 different drugs so that the polymer contains a functionalgroup that can react with a functional group of the NaPi2b-targetedantibody; and (6) reacting the modified polymer-drug conjugate of step(5) with the isolated NaPi2b-targeted antibody or its derivative to formthe conjugate disclosed herein. Steps (5) and (6) may be repeated if 2different isolated NaPi2b-targeted antibodies or their derivatives areto be conjugated to form a polymer-drug conjugate comprising twodifferent drugs and two different antibodies.

In yet another embodiment, the conjugates are formed in several steps.These steps include (1) modifying a polymer so that it contains afunctional group that can react with a functional group of the drug orits derivative; (2) further modifying the polymer so that it alsocontains a functional group that can react with a functional group ofthe NaPi2b-targeted antibody; (3) reacting the modified polymer with thedrug or its derivative so that the drug is linked to the polymer; and(4) reacting the modified polymer-drug conjugate with theNaPi2b-targeted antibody to form the conjugate disclosed herein. Thesequence of steps (1) and (2) or that of steps (3) and (4) can bereversed. Further either step (1) or (2) may be omitted if the modifiedpolymer contains a functional group that can react with both afunctional group of the drug or its derivatives and a functional groupof the NaPi2b-targeted antibody.

In another embodiment the conjugates are formed in several steps: (1)modifying a polymer so that it contains a functional group that canreact with a functional group of a first drug or its derivative; (2)further modifying a polymer so that it contains a functional group thatcan react with a functional group of the NaPi2b-targeted antibody; (3)reacting the modified polymer with the first drug or its derivative sothat the first drug is linked to the polymer; (4) modifying thepolymer-drug conjugate so that it contains a different functional groupthat can react with a functional group of a second drug or itsderivative (5) reacting the modified polymer-drug conjugate with thesecond drug or its derivative so that the second drug is linked to thepolymer-drug conjugate; (6) reacting the modified polymer-drug conjugatecontaining 2 different drugs so that the polymer with the isolatedNaPi2b-targeted antibody or its derivative to form the conjugatedisclosed herein. Step (6) may be repeated if 2 different isolatedantibodies or their derivatives are to be conjugated to form apolymer-drug conjugate comprising two different drugs and two differentantibodies. Step (4) may be carried out after step (1) so that themodified polymer contains two different functional groups that can reactwith two different drugs or their derivatives. In this embodiment, themodified polymer containing two different functional group that canreact with two different drugs or their derivatives can be furthermodified so that it contains a functional group that can react with afunctional group of the antibody; prior to the reaction of the modifiedpolymer with either the two different drugs (step (3) and step (5) orantibody (step (6).

In certain exemplary embodiments, the conjugates disclosed herein finduse in biomedical applications, such as drug delivery and tissueengineering, and the polymeric carrier is biocompatible andbiodegradable. In certain embodiments, the carrier is a soluble polymer,nanoparticle, gel, liposome, micelle, suture, implant, etc. In certainembodiments, the term “soluble polymer” encompasses biodegradablebiocompatible polymer such as a polyal (e.g., hydrophilic polyacetal orpolyketal). In certain other embodiments, the carrier is a fullysynthetic, semi-synthetic or naturally-occurring polymer. In certainother embodiments, the carrier is hydrophilic. Examples of suitablepolymeric carrier for producing the conjugates disclosed herein aredescribed in U.S. Pat. No. 8,815,226, the content of which is herebyincorporated by reference in its entirety.

In one embodiment, the polymeric carrier comprises units of Formula(IV):

wherein X′ indicates the substituent for the hydroxyl group of thepolymer backbone. As shown in Formula (IV) and the other formulaedescribed herein, each polyacetal unit has a single hydroxyl groupattached to the glycerol moiety of the unit and an X′ group attached tothe glycolaldehyde moiety of the unit. This is for convenience only andit should be construed that the polymer having units of Formula (IV) andother formulae described herein can contain a random distribution ofunits having a X′ group (or another substituent such as a linkercomprising a maleimide terminus) attached to the glycolaldehyde moietyof the units and those having a single X′ group (or another substituentsuch as a linker comprising a maleimide terminus) attached to theglycerol moiety of the units as well as units having two X′ groups (orother substituents such as a linker comprising a maleimide terminus)with one attached to the glycolaldehyde moiety and the other attached tothe glycerol moiety of the units.

In one embodiment, biodegradable biocompatible polyals suitable forpracticing the present disclosure have a molecular weight of betweenabout 2 and about 40 kDa, between about 6 and about 20 kDa, or betweenabout 8 and about 15 kDa). For example, the biodegradable biocompatiblepolyal used for the polymer scaffold or conjugate disclosed herein isPHF having a molecular weight of between about 2 and about 40 kDa (e.g.,about 2-20 kDa, 3-15 kDa, or 5-10 kDa.)

Methods for preparing polymer carriers (e.g., biocompatible,biodegradable polymer carriers) suitable for conjugation to modifiersare known in the art. For example, synthetic guidance can be found inU.S. Pat. Nos. 5,811,510; 5,863,990; 5,958,398; 7,838,619; 7,790,150;and 8,685,383. The skilled practitioner will know how to adapt thesemethods to make polymer carriers for use in the practice of thedisclosure.

In one embodiment, a method for forming the biodegradable biocompatiblepolyal conjugates of the present disclosure comprises a process by whicha suitable polysaccharide is combined with an efficient amount of aglycol-specific oxidizing agent to form an aldehyde intermediate. Thealdehyde intermediate, which is a polyal itself, may then be reduced tothe corresponding polyol, succinylated, and coupled with one or moresuitable modifiers to form a biodegradable biocompatible polyalconjugate comprising succinamide-containing linkages.

In another preferred embodiment, fully synthetic biodegradablebiocompatible polyals for used in the present disclosure can be preparedby reacting a suitable initiator with a suitable precursor compound.

For example, fully synthetic polyals may be prepared by condensation ofvinyl ethers with protected substituted diols. Other methods, such ascycle opening polymerization, may be used, in which the method efficacymay depend on the degree of substitution and bulkiness of the protectivegroups.

One of ordinary skill in the art will appreciate that solvent systems,catalysts and other factors may be optimized to obtain high molecularweight products.

In certain embodiments, the carrier is PHF.

In embodiments, the polymer carrier is PHF having a polydispersity index(PDI) of ≤1.5, e.g., <1.4, <1.3, <1.2 or <1.1.

For example, for conjugating the isolated NaPi2b-targeted antibodyhaving a molecular weight of 40 kDa to 200 kDa, the polymeric carrier ofthe scaffold is a polyacetal, e.g., a PHF having a molecular weight(i.e., MW of the unmodified PHF) ranging from about 2 kDa to about 40kDa (e.g., about 2-20 kDa, or about 3-15 kDa, or about 5-10 kDa).

For example, for conjugating the antibody having a molecular weight of40 kDa to 80 kDa, the polymeric carrier of the scaffold disclosed hereinis a polyacetal, e.g., a PHF having a molecular weight (i.e., MW of theunmodified PHF) ranging from about 2 kDa to about 40 kDa (e.g., about2-20 kDa, or about 3-15 kDa, or about 5-10 kDa). For example the PHF hasa molecular weight of about 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa,11 kDa, 12 kDa, 13 kDa, 14 kDa, or 15 kDa.

For example, for conjugating the antibody having a molecular weight of60 kDa to 120 kDa, the polymeric carrier of the scaffold disclosedherein is a polyacetal, e.g., a PHF having a molecular weight (i.e., MWof the unmodified PHF) ranging from about 2 kDa to about 40 kDa (e.g.,about 2-20 kDa, or about 3-15 kDa, or about 5-10 kDa). For example thePHF has a molecular weight of about 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa,10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, or 15 kDa.

For example, for conjugating the antibody having a molecular weight of140 kDa to 180 kDa or of 140 kDa to 150 kDa, the polymeric carrier ofthe scaffold disclosed herein is a polyacetal, e.g., a PHF having amolecular weight (i.e., MW of the unmodified PHF) ranging from about 2kDa to about 40 kDa (e.g., about 2-20 kDa, or about 3-15 kDa, or about5-10 kDa). For example the PHF has a molecular weight of about 5 kDa, 6kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, or 15kDa.

The antibody thereof in this molecular weight range, includes but arenot limited to, for example, full length antibodies, such as, IgG, IgM.

The biodegradable biocompatible conjugates disclosed herein can beprepared to meet desired requirements of biodegradability andhydrophilicity. For example, under physiological conditions, a balancebetween biodegradability and stability can be reached. For instance, itis known that molecules with molecular weights beyond a certainthreshold (generally, above 40-100 kDa, depending on the physical shapeof the molecule) are not excreted through kidneys, as small moleculesare, and can be cleared from the body only through uptake by cells anddegradation in intracellular compartments, most notably lysosomes. Thisobservation exemplifies how functionally stable yet biodegradablematerials may be designed by modulating their stability under generalphysiological conditions (pH=7.5±0.5) and at lysosomal pH (pH near 5).For example, hydrolysis of acetal and ketal groups is known to becatalyzed by acids, therefore polyals will be in general less stable inacidic lysosomal environment than, for example, in blood plasma. One candesign a test to compare polymer degradation profile at, for example,pH=5 and pH=7.5 at 37° C. in aqueous media, and thus to determine theexpected balance of polymer stability in normal physiologicalenvironment and in the “digestive” lysosomal compartment after uptake bycells. Polymer integrity in such tests can be measured, for example, bysize exclusion HPLC. One skilled on the art can select other suitablemethods for studying various fragments of the degraded conjugatesdisclosed herein.

In many cases, it will be preferable that at pH=7.5 the effective sizeof the polymer will not detectably change over 1 to 7 days, and remainwithin 50% from the original for at least several weeks. At pH=5, on theother hand, the polymer should preferably detectably degrade over 1 to 5days, and be completely transformed into low molecular weight fragmentswithin a two-week to several-month time frame. Although fasterdegradation may be in some cases preferable, in general it may be moredesirable that the polymer degrades in cells with the rate that does notexceed the rate of metabolization or excretion of polymer fragments bythe cells. Accordingly, in certain embodiments, the conjugates of thepresent disclosure are expected to be biodegradable, in particular uponuptake by cells, and relatively “inert” in relation to biologicalsystems. The products of carrier degradation are preferably unchargedand do not significantly shift the pH of the environment. It is proposedthat the abundance of alcohol groups may provide low rate of polymerrecognition by cell receptors, particularly of phagocytes. The polymerbackbones of the present disclosure generally contain few, if any,antigenic determinants (characteristic, for example, for somepolysaccharides and polypeptides) and generally do not comprise rigidstructures capable of engaging in “key-and-lock” type interactions invivo unless the latter are desirable. Thus, the soluble, crosslinked andsolid conjugates disclosed herein are predicted to have low toxicity andbioadhesivity, which makes them suitable for several biomedicalapplications.

In certain embodiments of the present disclosure, the biodegradablebiocompatible conjugates can form linear or branched structures. Forexample, the biodegradable biocompatible polyal conjugates of thepresent disclosure can be chiral (optically active). Optionally, thebiodegradable biocompatible polyal conjugates of the present disclosurecan be scalemic.

In certain embodiments, the conjugates disclosed herein arewater-soluble. In certain embodiments, the conjugates disclosed hereinare water-insoluble. In certain embodiments, the inventive conjugate isin a solid form. In certain embodiments, the conjugates disclosed hereinare colloids. In certain embodiments, the conjugates disclosed hereinare in particle form. In certain embodiments, the conjugates disclosedherein are in gel form.

Scheme 1 below shows a synthetic scheme of making a polymeric drugscaffold disclosed herein. In one embodiment, the conjugates are formedin several steps: (1) the polymer, PHF is modified to contain a COOHmoiety (e.g., —C(O)—X—(CH₂)₂—COOH); (2) the polymer is then furthermodified so that it contains a maleimido moiety (e.g., EG2-MI) that canreact with a functional group of a PBRM; (3) the modified polymer,containing two different functional groups, is reacted with a functionalgroup of a drug or its derivative (e.g., AF-HPA-Ala) to form apolymer-drug conjugate; (4) the disulfide bonds of a PBRM are reduced;(5) the reduced PBRM is then reacted with the polymer-drug conjugate toform the protein-polymer-drug conjugate; and (6) the remaining maleimidomoieties are optionally reacted with a maleimido blocking compound(e.g., cysteine).

In another embodiment the order of steps (2) and (3) can be reversed asdepicted in the right side route in Scheme 1 below.

In yet another embodiment, steps (2) and (3) above are carried outsimultaneously as depicted in Scheme 2 below.

Use of NaPi2b-Targeted Antibody-Drug Conjugates

It will be appreciated that administration of therapeutic entities inaccordance with the disclosure will be administered with suitablecarriers, excipients, and other agents that are incorporated intoformulations to provide improved transfer, delivery, tolerance, and thelike. A multitude of appropriate formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences (15th ed., Mack Publishing Company, Easton, Pa.(1975)), particularly Chapter 87 by Blaug, Seymour, therein. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as Lipofectin™), DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. Any of the foregoingmixtures may be appropriate in treatments and therapies in accordancewith the present disclosure, provided that the active ingredient in theformulation is not inactivated by the formulation and the formulation isphysiologically compatible and tolerable with the route ofadministration. See also Baldrick P. “Pharmaceutical excipientdevelopment: the need for preclinical guidance.” Regul. ToxicolPharmacol. 32(2):210-8 (2000), Wang W. “Lyophilization and developmentof solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2):1-60 (2000),Charman WN “Lipids, lipophilic drugs, and oral drug delivery-someemerging concepts.” J Pharm Sci. 89(8):967-78 (2000), Powell et al.“Compendium of excipients for parenteral formulations” PDA J Pharm SciTechnol. 52:238-311 (1998) and the citations therein for additionalinformation related to formulations, excipients and carriers well knownto pharmaceutical chemists.

In one embodiment, the NaPi2b antibody conjugates disclosed herein maybe used as therapeutic agents. Such agents will generally be employed todiagnose, prognose, monitor, treat, alleviate, prevent, and/or delay theprogression of a disease or pathology associated with, e.g., an aberrantNaPi2b activity and/or expression in a subject. A therapeutic regimen iscarried out by identifying a subject, e.g., a human patient sufferingfrom (or at risk of developing) a disease or disorder associated withaberrant NaPi2b activity and/or expression, e.g., a cancer, usingstandard methods. NaPi2b antibody conjugate preparation, preferably onehaving high specificity and high affinity for its target antigen, isadministered to the subject and will generally have an effect due to itsbinding with the target. Administration of the conjugate may abrogate orinhibit or interfere with the signaling function of the target.Administration of the conjugate may abrogate or inhibit or interferewith the binding of the target with an endogenous ligand to which itnaturally binds. For example, the conjugate can bind to the target andmodulate, block, inhibit, reduce, antagonize, neutralize, or otherwiseinterfere with NaPi2b activity and/or expression.

Diseases or disorders related to aberrant NaPi2b activity and/orexpression include but not limited to cancer. The target cancer can beovarian cancer such as epithelial ovarian cancer, thyroid cancer,colorectal cancer, lung cancer, non-small cell lung cancer (NSCLC) suchas non-squamous NSCLC, breast cancer, kidney cancer and salivary ductcarcinoma.

In another aspect, diseases or disorders are cancer selected from thegroup consisting of non-small cell lung cancer (NSCLC) such asnon-squamous NSCLC and ovarian cancer such as epithelial ovarian cancer.Generally, alleviation or treatment of a disease or disorder involvesthe lessening of one or more symptoms or medical problems associatedwith the disease or disorder. For example, in the case of cancer, thetherapeutically effective amount of the conjugate or a pharmaceuticalcomposition thereof can accomplish one or a combination of thefollowing: reduce the number of cancer cells; reduce the tumor size;inhibit (i.e., to decrease to some extent and/or stop) cancer cellinfiltration into peripheral organs; inhibit tumor metastasis; inhibit,to some extent, tumor growth; and/or relieve to some extent one or moreof the symptoms associated with the cancer. In some embodiments, acomposition disclosed herein can be used to prevent the onset orreoccurrence of the disease or disorder in a subject.

A therapeutically effective amount of a NaPi2b antibody conjugatedisclosed herein relates generally to the amount needed to achieve atherapeutic objective. As noted above, this may be a binding interactionbetween the antibody and its target antigen that, in certain cases,interferes with the functioning of the target. The amount required to beadministered will furthermore depend on the binding affinity of theantibody for its specific antigen, and will also depend on the rate atwhich an administered antibody is depleted from the free volume othersubject to which it is administered. The dosage regimen utilizing theconjugates disclosed herein is also selected in accordance with avariety of other factors including type, species, age, weight, sex andmedical condition of the patient; the severity of the condition to betreated; the route of administration; the renal and hepatic function ofthe patient; and the particular conjugate employed. An ordinarilyskilled physician or veterinarian can readily determine and prescribethe effective amount of the conjugate required to prevent, counter orarrest the progress of the condition. Common ranges for therapeuticallyeffective dosing of a NaPi2b antibody conjugate disclosed herein may be,by way of nonlimiting example, from about 0.1 mg/kg body weight to about50 mg/kg body weight, from about 0.1 mg/kg body weight to about 100mg/kg body weight or from about 0.1 mg/kg body weight to about 150 mg/kgbody weight. Ranges disclosed herein are expressed as amountadministered based on the subject's weight, and one skilled in the artcan easily express it as amount administered per body surface area ofthe subject. For example, 1 mg/kg body weight for a human adult isequivalent to about 37 mg/m² and 1 mg/kg body weight for a human childis equivalent to about 25 mg/m².

Common dosing frequencies may range, for example, from twice daily toonce a month (e.g., once daily, once weekly; once every other week; onceevery 3 weeks or monthly). For example, conjugates of XMT 1535 or10H1.11.4B disclosed herein can be administered (e.g., as a single doseweekly, every 2 weeks, every 3 weeks, or monthly) at about 0.1 mg/kg toabout 20 mg/kg (e.g., 0.2 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.67 mg/kg, 0.8mg/kg, 1 mg/kg, 1.25 mg/kg, 1.67 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 4mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg,12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19mg/kg, or 20 mg/kg). For example, conjugates of XMT 1535 or 10H1.11.4Bdisclosed herein can be administered (e.g., as a single dose weekly,every 2 weeks, every 3 weeks, or monthly) at about 0.1 mg/kg to about 20mg/kg (e.g., 0.2 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.67 mg/kg, 0.8 mg/kg, 1mg/kg, 1.25 mg/kg, 1.67 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 4 mg/kg, 5mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg,13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, or20 mg/kg) for treating NaPi2b-expressing ovarian cancer orNaPi2b-expressing NSCLC cancer.

Efficaciousness of treatment is determined in association with any knownmethod for diagnosing or treating the particular NaPi2b-relateddisorder. Alleviation of one or more symptoms of the NaPi2b-relateddisorder indicates that the antibody confers a clinical benefit.

Methods for the screening of antibodies that possess the desiredspecificity include, but are not limited to, enzyme linked immunosorbentassay (ELISA) and other immunologically mediated techniques known withinthe art.

Therapeutic Administration and Formulations of NaPi2b-TargetedAntibody-Drug Conjugates

The conjugates disclosed herein (also referred to herein as “activecompounds”), can be incorporated into pharmaceutical compositionssuitable for administration. Principles and considerations involved inpreparing such compositions, as well as guidance in the choice ofcomponents are provided, for example, in Remington's PharmaceuticalSciences: The Science And Practice Of Pharmacy 19th ed. (Alfonso R.Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.: 1995; DrugAbsorption Enhancement: Concepts, Possibilities, Limitations, AndTrends, Harwood Academic Publishers, Langhorne, Pa., 1994; and PeptideAnd Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4),1991, M. Dekker, New York.

Such compositions typically comprise the antibody and/or conjugatesthereof and a pharmaceutically acceptable carrier.

As used herein, the phrase “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, carriers, and/or dosage forms whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of human beings and animals without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

As used herein, the term “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration.Suitable carriers are described in the most recent edition ofRemington's Pharmaceutical Sciences, a standard reference text in thefield, which is incorporated herein by reference. Preferred examples ofsuch carriers or diluents include, but are not limited to, water,saline, ringer's solutions, dextrose solution, and 5% human serumalbumin. Liposomes and non-aqueous vehicles such as fixed oils may alsobe used. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe compositions is contemplated.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

A pharmaceutical composition disclosed herein is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid(EDTA); buffers such as acetates, citrates or phosphates, and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

In one embodiment, the pharmaceutical composition is in bulk or in unitdosage form. The unit dosage form is any of a variety of forms,including, for example, a capsule, an IV bag, a tablet, a single pump onan aerosol inhaler or a vial. The quantity of active ingredient (e.g., aconjugate disclosed herein) in a unit dose of composition is aneffective amount and is varied according to the particular treatmentinvolved. One skilled in the art will appreciate that it is sometimesnecessary to make routine variations to the dosage depending on the ageand condition of the patient. The dosage will also depend on the routeof administration.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL′ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser thatcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a sustained/controlled release formulations, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art.

For example, the active ingredients can be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacrylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions.

Sustained-release preparations can be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

The materials can also be obtained commercially from Alza Corporationand Nova Pharmaceuticals, Inc. Liposomal suspensions (includingliposomes targeted to infected cells with monoclonal antibodies to viralantigens) and can also be used as pharmaceutically acceptable carriers.These can be prepared according to methods known to those skilled in theart, for example, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms disclosed herein are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The formulation can also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Alternatively, or in addition, the composition can comprise an agentthat enhances its function, such as, for example, a cytotoxic agent,cytokine, chemotherapeutic agent, or growth-inhibitory agent. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended.

In one embodiment, the active compounds are administered in combinationtherapy, i.e., combined with other agents, e.g., therapeutic agents,that are useful for treating pathological conditions or disorders, suchas various forms of cancer, autoimmune disorders and inflammatorydiseases. The term “in combination” in this context means that theagents are given substantially contemporaneously, either simultaneouslyor sequentially. If given sequentially, at the onset of administrationof the second compound, the first of the two compounds is preferablystill detectable at effective concentrations at the site of treatment.

For example, the combination therapy can include one or more conjugatesdisclosed herein coformulated with, and/or coadministered with, one ormore additional antibodies e.g., a NaPi2b antibody, which can be thesame as the antibody used to form the conjugate or a different antibody.

For example, the combination therapy can include one or more therapeuticagent and/or adjuvant. In certain embodiments, the additionaltherapeutic agent is a small molecule inhibitor, another antibody-basedtherapy, a polypeptide or peptide-based therapy, a nucleic acid-basedtherapy and/or other biologic.

In certain embodiments, the additional therapeutic agent is a cytotoxicagent, a chemotherapeutic agent, a growth inhibitory agent, anangiogenesis inhibitor, a PARP (poly(ADP)-ribose polymerase) inhibitor,an alkylating agent, an anti-metabolite, an anti-microtubule agent, atopoisomerase inhibitor, a cytotoxic antibiotic, any other nucleic aciddamaging agent or an immune checkpoint inhibitor. In one embodiment, thetherapeutic agent used in the treatment of cancer, includes but is notlimited to, a platinum compound (e.g., cisplatin or carboplatin); ataxane (e.g., paclitaxel or docetaxel); a topoisomerase inhibitor (e.g.,irinotecan or topotecan); an anthracycline (e.g., doxorubicin(ADRIAMYCIN®) or liposomal doxorubicin (DOXIL®)); an anti-metabolite(e.g., gemcitabine, pemetrexed); cyclophosphamide; vinorelbine(NAVELBINE®); hexamethylmelamine; ifosfamide; etoposide; an angiogenesisinhibitor (e.g., Bevacizumab (Avastin®)), thalidomide, TNP-470, plateletfactor 4, interferon or endostatin); a PARP inhibitor (e.g., Olaparib(Lynparza™)); an immune checkpoint inhibitor, such as for example, amonoclonal antibody that targets either PD-1 or PD-L ((Pembrolizumab(Keytruda®)), atezolizumab (MPDL3280A) or Nivolumab (Opdivo®)) or CTA-4(Ipilimumab (Yervoy®), a kinase inhibitor (e.g., sorafenib orerlotinib), a proteasome inhibitor (e.g., bortezomib or carfilzomib), animmune modulating agent (e.g., lenalidomide or IL-2), a radiation agent,an ALK inhibitor (e.g. crizotinib (Xalkori), ceritinib (Zykadia),alectinib (Alecensa), dalantercept (ACE-041), brigatinib (AP26113),entrectinib (NMS-E628), PF-06463922 TSR-011, CEP-37440 and X-396) and/ora biosimilar thereof and/or combinations thereof. Other suitable agentsinclude an agent considered standard of care by those skilled in the artand/or a chemotherapeutic agent well known to those skilled in the art.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofCTLA-4. In some embodiments, the immune checkpoint inhibitor is anantibody against CTLA-4. In some embodiments, the immune checkpointinhibitor is a monoclonal antibody against CTLA-4. In other embodiments,the immune checkpoint inhibitor is a human or humanized antibody againstCTLA-4. In one embodiment, the anti-CTLA-4 antibody blocks the bindingof CTLA-4 to CD80 (B7-1) and/or CD86 (B7-2) expressed on antigenpresenting cells. Exemplary antibodies against CTLA-4 include, but arenot limited to, Bristol Meyers Squibb's anti-CTLA-4 antibody ipilimumab(also known as Yervoy®, MDX-010, BMS-734016 and MDX-101); anti-CTLA4Antibody, clone 9H10 from Millipore; Pfizer's tremelimumab (CP-675,206,ticilimumab); and anti-CTLA4 antibody clone BNI3 from Abcam.

In some embodiments, the anti-CTLA-4 antibody is an anti-CTLA-4 antibodydisclosed in any of the following patent publications (hereinincorporated by reference): WO 2001014424; WO 2004035607;US2005/0201994; EP 1212422 B1; WO2003086459; WO2012120125; WO2000037504;WO2009100140; W0200609649; WO2005092380; WO2007123737; WO2006029219;WO20100979597; W0200612168; and WO1997020574. Additional CTLA-4antibodies are described in U.S. Pat. Nos. 5,811,097, 5,855,887,6,051,227, and 6,984,720; in PCT Publication Nos. WO 01/14424 and WO00/37504; and in U.S. Publication Nos. 2002/0039581 and 2002/086014;and/or U.S. Pat. Nos. 5,977,318, 6,682,736, 7,109,003, and 7,132,281,incorporated herein by reference). In some embodiments, the anti-CTLA-4antibody is for example, those disclosed in: WO 98/42752; U.S. Pat. Nos.6,682,736 and 6,207,156; Hurwitz et al, Proc. Natl. Acad. Sci. USA,95(17): 10067-10071 (1998); Camacho et al, J. Clin. Oncol., 22(145):Abstract No. 2505 (2004) (antibody CP-675206); Mokyr et al, Cancer Res.,58:5301-5304 (1998) (incorporated herein by reference).

In some embodiments, the CTLA-4 inhibitor is a CTLA-4 ligand asdisclosed in WO1996040915.

In some embodiments, the CTLA-4 inhibitor is a nucleic acid inhibitor ofCTLA-4 expression. For example, anti-CTLA4 RNAi molecules may take theform of the molecules described by Mello and Fire in PCT PublicationNos. WO 1999/032619 and WO 2001/029058; U.S. Publication Nos.2003/0051263, 2003/0055020, 2003/0056235, 2004/265839, 2005/0100913,2006/0024798, 2008/0050342, 2008/0081373, 2008/0248576, and 2008/055443;and/or U.S. Pat. Nos. 6,506,559, 7,282,564, 7,538,095, and 7,560,438(incorporated herein by reference). In some instances, the anti-CTLA4RNAi molecules take the form of double stranded RNAi molecules describedby Tuschl in European Patent No. EP 1309726 (incorporated herein byreference). In some instances, the anti-CTLA4 RNAi molecules take theform of double stranded RNAi molecules described by Tuschl in U.S. Pat.Nos. 7,056,704 and 7,078,196 (incorporated herein by reference). In someembodiments, the CTLA4 inhibitor is an aptamer described in PCTPublication No. WO2004081021.

Additionally, the anti-CTLA4 RNAi molecules of the present disclosuremay take the form be RNA molecules described by Crooke in U.S. Pat. Nos.5,898,031, 6,107,094, 7,432,249, and 7,432,250, and European ApplicationNo. EP 0928290 (incorporated herein by reference).

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofPD-L1. In some embodiments, the immune checkpoint inhibitor is anantibody against PD-L1. In some embodiments, the immune checkpointinhibitor is a monoclonal antibody against PD-L1. In other or additionalembodiments, the immune checkpoint inhibitor is a human or humanizedantibody against PD-L1. In one embodiment, the immune checkpointinhibitor reduces the expression or activity of one or more immunecheckpoint proteins, such as PD-L1. In another embodiment, the immunecheckpoint inhibitor reduces the interaction between PD-1 and PD-L1.Exemplary immune checkpoint inhibitors include antibodies (e.g., ananti-PD-L1 antibody), RNAi molecules (e.g., anti-PD-L1 RNAi), antisensemolecules (e.g., an anti-PD-L1 antisense RNA), dominant negativeproteins (e.g., a dominant negative PD-L1 protein), and small moleculeinhibitors. Antibodies include monoclonal antibodies, humanizedantibodies, deimmunized antibodies, and Ig fusion proteins. An exemplaryanti-PD-L1 antibody includes clone EH12. Exemplary antibodies againstPD-L1 include: Genentech's MPDL3280A (RG7446); Anti-mouse PD-L1 antibodyClone 10F.9G2 (Cat #BE0101) from BioXcell; anti-PD-L1 monoclonalantibody MDX-1105 (BMS-936559) and BMS-935559 from Bristol-Meyer'sSquibb; MSB0010718C; mouse anti-PD-L1 Clone 29E.2A3; and AstraZeneca'sMEDI4736. In some embodiments, the anti-PD-L1 antibody is an anti-PD-L1antibody disclosed in any of the following patent publications (hereinincorporated by reference): WO2013079174; CN101104640; WO2010036959;WO2013056716; WO2007005874; WO2010089411; WO2010077634; WO2004004771;WO2006133396; W0201309906; US 20140294898; WO2013181634 or WO2012145493.

In some embodiments, the PD-L1 inhibitor is a nucleic acid inhibitor ofPD-L1 expression. In some embodiments, the PD-L1 inhibitor is disclosedin one of the following patent publications (incorporated herein byreference): WO2011127180 or WO2011000841. In some embodiments, the PD-L1inhibitor is rapamycin.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofPD-L2. In some embodiments, the immune checkpoint inhibitor is anantibody against PD-L2. In some embodiments, the immune checkpointinhibitor is a monoclonal antibody against PD-L2. In other or additionalembodiments, the immune checkpoint inhibitor is a human or humanizedantibody against PD-L2. In some embodiments, the immune checkpointinhibitor reduces the expression or activity of one or more immunecheckpoint proteins, such as PD-L2. In other embodiments, the immunecheckpoint inhibitor reduces the interaction between PD-1 and PD-L2.Exemplary immune checkpoint inhibitors include antibodies (e.g., ananti-PD-L2 antibody), RNAi molecules (e.g., an anti-PD-L2 RNAi),antisense molecules (e.g., an anti-PD-L2 antisense RNA), dominantnegative proteins (e.g., a dominant negative PD-L2 protein), and smallmolecule inhibitors. Antibodies include monoclonal antibodies, humanizedantibodies, deimmunized antibodies, and Ig fusion proteins.

In some embodiments, the PD-L2 inhibitor is GlaxoSmithKline's AMP-224(Amplimmune). In some embodiments, the PD-L2 inhibitor is rHIgM12B7.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofPD-L1. In some embodiments, the immune checkpoint inhibitor is anantibody against PD-1. In some embodiments, the immune checkpointinhibitor is a monoclonal antibody against PD-1. In other embodiments,the immune checkpoint inhibitor is a human or humanized antibody againstPD-1. For example, the inhibitors of PD-1 biological activity (or itsligands) disclosed in U.S. Pat. Nos. 7,029,674; 6,808,710; or U.S.Patent Application Nos: 20050250106 and 20050159351 can be used in thecombinations provided herein. Exemplary antibodies against PD-1 include:Anti-mouse PD-1 antibody Clone J43 (Cat #BE0033-2) from BioXcell;Anti-mouse PD-1 antibody Clone RMP1-14 (Cat #BE0146) from BioXcell;mouse anti-PD-1 antibody Clone EH12; Merck's MK-3475 anti-mouse PD-1antibody (Keytruda®, pembrolizumab, lambrolizumab, h409A1 1); andAnaptysBio's anti-PD-1 antibody, known as ANB011; antibody MDX-1 106(ONO-4538); Bristol-Myers Squibb's human IgG4 monoclonal antibodynivolumab (Opdivo®, BMS-936558, MDX1106); AstraZeneca's AMP-514, andAMP-224; and Pidilizumab (CT-011 or hBAT-1), CureTech Ltd.

Additional exemplary anti-PD-1 antibodies are described by Goldberg etal, Blood 1 10(1): 186-192 (2007), Thompson et al, Clin. Cancer Res.13(6): 1757-1761 (2007), and Korman et al, International Application No.PCT/JP2006/309606 (publication no. WO 2006/121168 A1), each of which areexpressly incorporated by reference herein. In some embodiments, theanti-PD-1 antibody is an anti-PD-1 antibody disclosed in any of thefollowing patent publications (herein incorporated by reference):W0014557; WO2011110604; WO2008156712; US2012023752; WO2011110621;WO2004072286; WO2004056875; WO20100036959; WO2010029434; W0201213548;WO2002078731; WO2012145493; WO2010089411; WO2001014557; WO2013022091;WO2013019906; WO2003011911; US20140294898; and WO2010001617.

In some embodiments, the PD-1 inhibitor is a PD-1 binding protein asdisclosed in W0200914335 (herein incorporated by reference).

In some embodiments, the PD-1 inhibitor is a peptidomimetic inhibitor ofPD-1 as disclosed in WO2013132317 (herein incorporated by reference).

In some embodiments, the PD-1 inhibitor is an anti-mouse PD-1 mAb: cloneJ43, BioXCell (West Lebanon, N.H.).

In some embodiments, the PD-1 inhibitor is a PD-L1 protein, a PD-L2protein, or fragments, as well as antibody MDX-1 106 (ONO-4538) testedin clinical studies for the treatment of certain malignancies (Brahmeret al., J Clin Oncol. 2010 28(19): 3167-75, Epub 2010 Jun. 1). Otherblocking antibodies may be readily identified and prepared by theskilled person based on the known domain of interaction between PD-1 andPD-L1/PD-L2, as discussed above. For example, a peptide corresponding tothe IgV region of PD-1 or PD-L1/PD-L2 (or to a portion of this region)could be used as an antigen to develop blocking antibodies using methodswell known in the art.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofIDO1. In some embodiments, the immune checkpoint inhibitor is a smallmolecule against IDO1. Exemplary small molecules against IDO1 include:Incyte's INCB024360, NSC-721782 (also known as 1-methyl-D-tryptophan),and Bristol Meyers Squibb's F001287.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofLAG3 (CD223). In some embodiments, the immune checkpoint inhibitor is anantibody against LAG3. In some embodiments, the immune checkpointinhibitor is a monoclonal antibody against LAG3. In other or additionalembodiments, the immune checkpoint inhibitor is a human or humanizedantibody against LAG3. In additional embodiments, an antibody againstLAG3 blocks the interaction of LAG3 with major histocompatibilitycomplex (MHC) class II molecules. Exemplary antibodies against LAG3include: anti-Lag-3 antibody clone eBioC9B7W (C9B7W) from eBioscience;anti-Lag3 antibody LS-B2237 from LifeSpan Biosciences; IMP321 (ImmuFact)from Immutep; anti-Lag3 antibody BMS-986016; and the LAG-3 chimericantibody A9H12. In some embodiments, the anti-LAG3 antibody is ananti-LAG3 antibody disclosed in any of the following patent publications(herein incorporated by reference): WO2010019570; WO2008132601; orWO2004078928.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst TIM3 (also known as HAVCR2). In some embodiments, the immunecheckpoint inhibitor is a monoclonal antibody against TIM3. In other oradditional embodiments, the immune checkpoint inhibitor is a human orhumanized antibody against TIM3. In additional embodiments, an antibodyagainst TIM3 blocks the interaction of TIM3 with galectin-9 (Ga19). Insome embodiments, the anti-TIM3 antibody is an anti-TIM3 antibodydisclosed in any of the following patent publications (hereinincorporated by reference): WO2013006490; W0201155607; WO2011159877; orW0200117057. In another embodiment, a TIM3 inhibitor is a TIM3 inhibitordisclosed in WO2009052623.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst B7-H3. In one embodiment, the immune checkpoint inhibitor isMGA271.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst MR. In one embodiment, the immune checkpoint inhibitor isLirilumab (IPH2101). In some embodiments, an antibody against MR blocksthe interaction of KIR with HLA.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst CD137 (also known as 4-1BB or TNFRSF9). In one embodiment, theimmune checkpoint inhibitor is urelumab (BMS-663513, Bristol-MyersSquibb), PF-05082566 (anti-4-1BB, PF-2566, Pfizer), or XmAb-5592(Xencor). In one embodiment, an anti-CD137 antibody is an antibodydisclosed in U.S. Published Application No. US 2005/0095244; an antibodydisclosed in issued U.S. Pat. No. 7,288,638 (such as 20H4.9-IgG4 [1007or BMS-663513] or 20H4.9-IgG1 [BMS-663031]); an antibody disclosed inissued U.S. Pat. No. 6,887,673 [4E9 or BMS-554271]; an antibodydisclosed in issued U.S. Pat. No. 7,214,493; an antibody disclosed inissued U.S. Pat. No. 6,303,121; an antibody disclosed in issued U.S.Pat. No. 6,569,997; an antibody disclosed in issued U.S. Pat. No.6,905,685; an antibody disclosed in issued U.S. Pat. No. 6,355,476; anantibody disclosed in issued U.S. Pat. No. 6,362,325 [1D8 or BMS-469492;3H3 or BMS-469497; or 3E1]; an antibody disclosed in issued U.S. Pat.No. 6,974,863 (such as 53A2); or an antibody disclosed in issued U.S.Pat. No. 6,210,669 (such as 1D8, 3B8, or 3E1). In a further embodiment,the immune checkpoint inhibitor is one disclosed in WO 2014036412. Inanother embodiment, an antibody against CD137 blocks the interaction ofCD137 with CD137L.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst PS. In one embodiment, the immune checkpoint inhibitor isBavituximab.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst CD52. In one embodiment, the immune checkpoint inhibitor isalemtuzumab.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst CD30. In one embodiment, the immune checkpoint inhibitor isbrentuximab vedotin. In another embodiment, an antibody against CD30blocks the interaction of CD30 with CD30L.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst CD33. In one embodiment, the immune checkpoint inhibitor isgemtuzumab ozogamicin.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst CD20. In one embodiment, the immune checkpoint inhibitor isibritumomab tiuxetan. In another embodiment, the immune checkpointinhibitor is ofatumumab. In another embodiment, the immune checkpointinhibitor is rituximab. In another embodiment, the immune checkpointinhibitor is tositumomab.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst CD27 (also known as TNFRSF7). In one embodiment, the immunecheckpoint inhibitor is CDX-1127 (Celldex Therapeutics). In anotherembodiment, an antibody against CD27 blocks the interaction of CD27 withCD70.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst OX40 (also known as TNFRSF4 or CD134). In one embodiment, theimmune checkpoint inhibitor is anti-OX40 mouse IgG. In anotherembodiment, an antibody against 0×40 blocks the interaction of OX40 withOX40L.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst glucocorticoid-induced tumor necrosis factor receptor (GITR). Inone embodiment, the immune checkpoint inhibitor is TRX518 (GITR, Inc.).In another embodiment, an antibody against GITR blocks the interactionof GITR with GITRL.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst inducible T-cell COStimulator (ICOS, also known as CD278). Inone embodiment, the immune checkpoint inhibitor is MEDI570 (MedImmune,LLC) or AMG557 (Amgen). In another embodiment, an antibody against ICOSblocks the interaction of ICOS with ICOSL and/or B7-H2.

In some embodiments, the immune checkpoint inhibitor is an inhibitoragainst BTLA (CD272), CD160, 2B4, LAIR1, TIGHT, LIGHT, DR3, CD226, CD2,or SLAM. As described elsewhere herein, an immune checkpoint inhibitorcan be one or more binding proteins, antibodies (or fragments orvariants thereof) that bind to immune checkpoint molecules, nucleicacids that downregulate expression of the immune checkpoint molecules,or any other molecules that bind to immune checkpoint molecules (i.e.small organic molecules, peptidomimetics, aptamers, etc.). In someinstances, an inhibitor of BTLA (CD272) is HVEM. In some instances, aninhibitor of CD160 is HVEM. In some cases, an inhibitor of 2B4 is CD48.In some instances, an inhibitor of LAIR1 is collagen. In some instances,an inhibitor of TIGHT is CD112, CD113, or CD155. In some instances, aninhibitor of CD28 is CD80 or CD86. In some instances, an inhibitor ofLIGHT is HVEM. In some instances, an inhibitor of DR3 is TL1A. In someinstances, an inhibitor of CD226 is CD155 or CD112. In some cases, aninhibitor of CD2 is CD48 or CD58. In some cases, SLAM is self inhibitoryand an inhibitor of SLAM is SLAM.

In certain embodiments, the immune checkpoint inhibitor inhibits acheckpoint protein that include, but are not limited to CTLA4 (cytotoxicT-lymphocyte antigen 4, also known as CD152), PD-L1 (programmed celldeath 1 ligand 1, also known as CD274), PDL2 programmed cell deathprotein 2), PD-1 (programmed cell death protein 1, also known as CD279),a B-7 family ligand (B7-H1, B7-H3, B7-H4) BTLA (B and T lymphocyteattenuator, also known as CD272), HVEM, TIM3 (T-cell membrane protein3), GAL9, LAG-3 (lymphocyte activation gene-3; CD223), VISTA, KIR(killer immunoglobulin receptor), 2B4 (also known as CD244), CD160,CGEN-15049, CHK1 (Checkpoint kinase 1), CHK2 (Checkpoint kinase 2), A2aR(adenosine A2a receptor), CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86,CD137, CD226, CD276, DR3, GITR, HAVCR2, HVEM, IDO1 (indoleamine2,3-dioxygenase 1), IDO2 (indoleamine 2,3-dioxygenase 2), ICOS(inducible T cell costimulator), LAIR1, LIGHT (also known as TNFSF14, aTNF family member), MARCO (macrophage receptor with collagenousstructure), OX40 (also known as tumor necrosis factor receptorsuperfamily, member 4, TNFRSF4, and CD134) and its ligand OX40L (CD252),SLAM, TIGHT, VTCN1 or a combination thereof.

In certain embodiments, the immune checkpoint inhibitor interacts with aligand of a checkpoint protein that comprises CTLA-4, PDL1, PDL2, PD1,BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1,CHK2, A2aR, a B-7 family ligand, CD2, CD27, CD28, CD30, CD40, CD70,CD80, CD86, CD137, CD226, CD276, DR3, GITR, HAVCR2, HVEM, IDO1, IDO2,ICOS (inducible T cell costimulator), LAIR1, LIGHT, MARCO (macrophagereceptor with collagenous structure), OX-40, SLAM, TIGHT, VTCN1 or acombination thereof.

In certain embodiments, the immune checkpoint inhibitor inhibits acheckpoint protein that comprises CTLA-4, PDL1, PD1 or a combinationthereof.

In certain embodiments, the immune checkpoint inhibitor inhibits acheckpoint protein that comprises CTLA-4 and PD1 or a combinationthereof.

In certain embodiments, the immune checkpoint inhibitor comprisespembrolizumab (MK-3475), nivolumab (BMS-936558), pidilizumab (CT-011),AMP-224, MDX-1 105, durvalumab (MEDI4736), MPDL3280A, BMS-936559,IPH2101, TSR-042, TSR-022, ipilimumab, lirilumab, atezolizumab,avelumab, tremelimumab, or a combination thereof.

In certain embodiments, the immune checkpoint inhibitor is nivolumab(BMS-936558), ipilimumab, pembrolizumab, atezolizumab, tremelimumab,durvalumab, avelumab, or a combination thereof.

In certain embodiments, the immune checkpoint inhibitor ispembrolizumab.

Diagnostic and Prophylactic Formulations

The NaPi2b antibody conjugates disclosed herein are used in diagnosticand prophylactic formulations. In one embodiment, a NaPi2b antibodyconjugate disclosed herein is administered to patients that are at riskof developing one or more of the aforementioned diseases, such as forexample, without limitation, cancer. A patient's or organ'spredisposition to one or more of the aforementioned indications can bedetermined using genotypic, serological or biochemical markers.

In another embodiment of the disclosure, a NaPi2b antibody conjugatedisclosed herein is administered to human individuals diagnosed with aclinical indication associated with one or more of the aforementioneddiseases, such as for example, without limitation, cancer. Upondiagnosis, a NaPi2b antibody conjugate disclosed herein is administeredto mitigate or reverse the effects of the clinical indication associatedwith one or more of the aforementioned diseases.

In another embodiment of the disclosure, a method for identifying anovarian cancer patient amenable to treatment with the conjugatesdisclosed herein, comprise measuring the status of certaincharacteristics in a tumor sample obtained from the patient, andidentifying the patient for treatment based on the status of certaincharacteristics in the tumor sample.

In another embodiment of the disclosure, a method for identifying NSCLCpatient amenable to treatment with the conjugates disclosed herein,comprise measuring the status of certain characteristics in a tumorsample obtained from the patient, and identifying the patient fortreatment based on the status of certain characteristics in the tumorsample.

Antibodies disclosed herein are also useful in the detection of NaPi2bin patient samples and accordingly are useful as diagnostics. Forexample, NaPi2b antibodies disclosed herein are used in in vitro assays,e.g., ELISA, to detect NaPi2b levels in a patient sample.

In one embodiment, a NaPi2b antibody disclosed herein is immobilized ona solid support (e.g., the well(s) of a microtiter plate). Theimmobilized antibody serves as a capture antibody for any NaPi2b thatmay be present in a test sample. Prior to contacting the immobilizedantibody with a patient sample, the solid support is rinsed and treatedwith a blocking agent such as milk protein or albumin to preventnonspecific adsorption of the analyte.

Subsequently the wells are treated with a test sample suspected ofcontaining the antigen, or with a solution containing a standard amountof the antigen. Such a sample is, e.g., a serum sample from a subjectsuspected of having levels of circulating antigen considered to bediagnostic of a pathology. After rinsing away the test sample orstandard, the solid support is treated with a second antibody that isdetectably labeled. The labeled second antibody serves as a detectingantibody. The level of detectable label is measured, and theconcentration of NaPi2b antigen in the test sample is determined bycomparison with a standard curve developed from the standard samples.

It will be appreciated that based on the results obtained using theNaPi2b antibodies disclosed herein in an in vitro diagnostic assay, itis possible to stage a disease in a subject based on expression levelsof the NaPi2b antigen. For a given disease, samples of blood are takenfrom subjects diagnosed as being at various stages in the progression ofthe disease, and/or at various points in the therapeutic treatment ofthe disease. Using a population of samples that provides statisticallysignificant results for each stage of progression or therapy, a range ofconcentrations of the antigen that may be considered characteristic ofeach stage is designated.

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same. The disclosurehaving now been described by way of written description, those of skillin the art will recognize that the disclosure can be practiced in avariety of embodiments and that the foregoing description and examplesbelow are for purposes of illustration and not limitation of the claimsthat follow.

EXAMPLES

The following working examples are illustrative of the linkers, drugmolecules and PBRM, and methods for preparing same. These are notintended to be limiting and it will be readily understood by one ofskill in the art that other reagents or methods may be utilized.

Abbreviations

The following abbreviations are used in the reaction schemes andsynthetic examples, which follow. This list is not meant to be anall-inclusive list of abbreviations used in the application asadditional standard abbreviations, which are readily understood by thoseskilled in the art of organic synthesis, can also be used in thesynthetic schemes and examples.

AF-HPA Auristatin F-hydroxypropylamide

FBS Fetal bovine serum

MMAE Monomethyl auristatin E

NaPi2b Type II sodium-phosphate co-transporter

NSCLC Non-small cell lung cancer

General Information

CDRs were identified by the Kabat numbering scheme.

Tumor growth inhibition (% TGI) was defined as the percent difference inmedian tumor volumes (MTVs) between treated and control groups.

Treatment efficacy was determined from the incidence and magnitude ofregression responses of the tumor size observed during the study.Treatment may cause partial regression (PR) or complete regression (CR)of the tumor in an animal. In a PR response, the tumor volume was 50% orless of its Day 1 volume for three consecutive measurements during thecourse of the study, and equal to or greater than 13.5 mm³ for one ormore of these three measurements. In a CR response, the tumor volume wasless than 13.5 mm3 for three consecutive measurements during the courseof the study. An animal with a CR response at the termination of a studywas additionally classified as a tumor-free survivor (TFS). Animals weremonitored for regression responses.

AF-HPA was prepared in a fashion similar to that as described in U.S.Pat. No. 8,685,383, Example 48.

Anti-NaPi2b antibody (XMT-1535) was produced using SEQ ID NO: 38 and SEQID NO: 39 from U.S. Pat. No. 8,603,474B2.

Anti-NaPi2b antibody (10H1.11.4B) was produced using SEQ ID NO: 80 andSEQ ID NO: 81 from U.S. Pat. No. 8,535,675B2.

HPLC purification was performed on a Phenomenex Gemini 5 μm 110 Å,250×10 mm, 5 micron, semi-preparation column.

SEC was performed on a Tosoh Biosciences TSK gel G4000 column (7.8 mm×30cm, 10 um) or Superose 12 column (GE Healthcare).

WCX was performed on ProPac WCX-10 (94 mm×250 mm) column (ThermoFisher).

Whenever possible the drug content of the conjugates was determinedspectrophotometrically otherwise LC/MS or ¹H-NMR was performed forquantitative determination of the drug content.

The protein content of the protein-polymer-drug conjugates wasdetermined spectrophotometrically at 280 nm or by ELISA.

The molecular weights of the polymer conjugates (reported as theapparent weight average molecular weights or peak molecular weights)were determined by SEC with either polysaccharide or protein molecularweight standards. More specifically, for the polymer or polymer-drugconjugates, polysaccharide molecular weights standard were used, and forprotein-drug-polymer conjugates, protein standards are used. Unlessspecifically indicated the reported polymer carrier molecular weight isthe weight average molecular weight of PHF; and the polymer-drugconjugate molecular weight and the protein-polymer-drug conjugates isthe peak molecular weight. The NaPi2b antibody-polymer-drug conjugateshave a peak molecular weight of about 170 kDa to about 250 kDa. Thepolymer and polymer conjugates synthesized/measured typically have apolydispersity ≤1.5.

NaPi2b antibody-polymer-drug conjugates were separated from residualunreacted drug-polymer conjugates by (Prep-WCX HPLC). If necessary,additional purification by size exclusion chromatography was conductedto remove any aggregated NaPi2b antibody-polymer-drug conjugates. Ingeneral, the NaPi2b antibody-polymer-drug conjugates typically contained<5% (w/w) aggregated fraction as determined by SEC; <0.5% (w/w, e.g.,<0.1% w/w) free (unconjugated) drug as determined by RP-HPLC orLC-MS/MS; <1% (w/w) of free polymer-drug conjugate as determined byRP-HPLC and <2% (w/w, e.g., <1% w/w) unconjugated NaPi2b as determinedby HIC-HPLC and/or WCX HPLC. Reduced or partially reduced antibodieswere prepared using procedures described in the literature, see, forexample, Francisco et al., Blood 102 (4): 1458-1465 (2003). The totaldrug (conjugated and unconjugated) concentration was determined byLC-MS/MS.

To determine the pharmacokinetics of the NaPi2b antibody-polymer-drugconjugates, assays were developed to measure the plasma concentration ofthe NaPi2b antibody-PHF-AF-HPA conjugate (i.e., conjugated AF-HPA) andconcentration of the released, unconjugated AF-HPA and AF (free drugs).To determine the plasma concentration of the free drugs, the acidifiedplasma sample was treated with acetonitrile to precipitate plasmaproteins and antibody drug conjugate and the acetonitrile containingsupernatant was analyzed for free drugs by LC-MS/MS. To determine theconcentration of conjugated AF-HPA, the acidified plasma sample wassubjected to basic hydrolysis followed by acidification and proteinprecipitation with acetonitrile. The acetonitrile supernatant containingthe released AF-HPA and AF was analyzed by LC-MS/MS. The standard curvesfor the free drugs and conjugated AF-HPA in plasma were linear over theconcentration ranges of 1 to 3,000 ng/mL and 10 to 20,000 ng/mL,respectively. Total NaPi2b concentration was determined by ELISA.

General Procedures

General Procedure A. Conjugation of Polymer with Linker or Drug

In general, the conjugation of the polymer (PHF-BA or PHF-GA) with anamine-containing linker, such as, for example, EG2-maleimide or anamine-containing linker drug, such as, for example, AF-HPA-Ala, HPA-Ala,is conducted in an aqueous or 10-90% organic/aqueous solvent mixture inthe presence of an activating agent, such as, for example EDC.HCl.Typical organic solvents, include, but are not limited to, watermiscible solvents, such as, for example, DMSO, DMF, DMA, NMP, propyleneglycol and ACN. To accelerate the coupling, a co-activator, such as, forexample, NHS, is added. The polymer is first mixed with theamino-containing compound followed by addition of the co-activator (NHS)and then the addition of the activator (EDC.HCl). The reaction isconducted at 0-10° C., pH 4.5 to 7.5 for 1 h to 24 hours at ambienttemperature. The resulting polymer conjugated product is purified bydiafiltration or by SEC. The product is concentrated to 2-50 mg/mL, thepH is adjusted to 4.5 to 6.5 to insure drug-polymer linker stability andthe conjugate is stored frozen at −20 to −80° C. until further use.

The conjugation of the polymer with the amine-containing linker or drugcan be conducted sequentially, in any order, or simultaneously.

General Procedure B. Partial Selective Reduction of Protein (NaPi2bAntibody)

The partial selective reduction of the inter-chain disulfide groups orunpaired disulfide in the relevant NaPi2b antibody prior to conjugationwith the polymer-drug conjugate is achieved by using a reducing agent,such as, for example, TCEP, DTT or β-mercaptoethanol. When the reductionis performed with an excess of the reducing agent, the reducing agent isremoved prior to conjugation by diafiltration or SEC. The degree ofconversion of the NaPi2b disulfide groups into reactive sulfhydrylgroups depends on the stoichiometry of NaPi2b, reducing agent, pH,temperature and/or duration of the reaction. When some but not all ofthe disulfide groups in the PBRM are reduced, the reduced PBRM is apartially reduced NaPi2b.

General Procedure C. Conjugation of Partially Reduced NaPi2b-TargetedAntibody with Polymer Drug Conjugate

The conjugation of the partially reduced NaPi2b-targeted antibody to thepolymer-drug conjugate is conducted under neutral or slightly basicconditions (pH 6.5-8.5) at antibody concentrations of 1-10 mg/mL andpolymer-drug conjugate concentration of 0.5-10 mg/mL. The polymer-drugconjugate is typically used in 1-5 fold excess relative to the desiredprotein-polymer-drug conjugate stoichiometry. When the antibody isconjugated to the maleimido group of the polymer-drug conjugate, theconjugation is optionally terminated by the addition of a water-solublemaleimido blocking compound, such as, for example, N-acetyl cysteine,cysteine methyl ester, N-methyl cysteine, 2-mercaptoethanol,3-mercaptopropanoic acid, 2-mercaptoacetic acid, mercaptomethanol (i.e.,HOCH₂SH), benzyl thiol, and the like.

The resulting NaPi2b-targeted antibody-polymer-drug conjugate istypically purified by diafiltration to remove any unconjugatedpolymer-drug conjugate, unconjugated drug and small molecule impurities.Alternatively or additionally, appropriate chromatographic separationprocedures such as, for example, size-exclusion chromatography,hydrophobic interaction chromatography, ion chromatography such as, forexample, WCX chromatography; reversed phase chromatography, hydroxylapatite chromatography, affinity chromatography or combination thereofmay be used to purify the NaPi2b antibody-polymer-drug conjugate. Theresulting purified NaPi2b-polymer-drug conjugate is typically formulatedin a buffer at pH 5.0-6.5.

Example 1: Synthesis of XMT-1535-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala)))

The XMT-1535-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) conjugates wereprepared using the procedure described in US Application PublicationUS-2015-0104407-A1. Table I gives the details of theantibody-polymer-drug conjugates.

TABLE I Example No. DAR (Drug:Antibody ratio) 1A About 8:1 to about 12:11B About 10:1 to about 14:1 1C About 11:1 to about 15:1

The XMT-1535-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) conjugates had a peakmolecular weight of about 180 kDa to about 250 kDa. The polymer andpolymer conjugates synthesized/measured typically have a polydispersity≤1.5. The polymer-drug conjugates (i.e., the drug-carrying polymer chainattached to antibody) contained about 25% mol to about 35% molbeta-alanine, about 7.0% mol to about 10% mol AF-HPA-Ala and about 1.5%mol to about 4% mol EG2-MI.

Example 2: Synthesis of (10H1.11.4B)-(EG2-MI-(10 kDaPHF-BA-(AF-HPA-Ala)))

The (10H11.1.4B)-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) conjugates(non-binding control) were prepared using the procedure described in USApplication Publication US-2015-0104407-A1. Table II gives the detailsof the antibody-polymer drug conjugates.

TABLE II Example No. DAR (Drug:Antibody ratio) 2A About 10:1 to about14:1 2B About 14:1 to about 19:1 2C About 9:1 to about 13:1

The (10H1.11.4B)-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) conjugates had apeak molecular weight of about 170 kDa to about 230 kDa. The polymer andpolymer conjugates synthesized/measured typically have a polydispersity≤1.5. The polymer-drug conjugates (i.e., the drug-carrying polymer chainattached to antibody) contained about 25% mol to about 35% molbeta-alanine, about 7% mol to about 10% mol AF-HPA-Ala and about 1.5%mol to about 4% mol EG2-MI.

Example 3: Synthesis of Rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala)))

The rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) conjugates(non-binding control) were prepared using the procedure described in USApplication Publication US-2015-0104407-A1. Table III gives the detailsof the antibody-polymer-drug conjugates.

TABLE III Example No. DAR (Drug:Antibody ratio) 3A About 15:1 to about21:1 3B About 14:1 to about 19:1

The rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) conjugates had apeak molecular weight of about 170 kDa to about 230 kDa. The polymer andpolymer conjugates synthesized/measured typically have a polydispersity≤1.5. The polymer-drug conjugates (i.e., the drug-carrying polymer chainattached to antibody) contained about 25% mol to about 35% molbeta-alanine, about 7% mol to about 10% mol AF-HPA-Ala and about 1.5%mol to about 4% mol EG2-MI.

Example 4: Synthesis of (10H1.11.4B)-(MC-VC-PABA-MMAE)

The (10H1.11.4B)-(maleimide -VC-PABA-MMAE) conjugates were preparedusing the procedure described in Doronina et al., Nature biotechnology,21: 778-784 (2003). Table IV gives the details of the antibody-drugconjugates.

TABLE IV DAR Example No. (Drug:Antibody ratio) 4A 4:1 4B 3:1

Example 5: Cytotoxicity Assays for NaPi2b-(EG2-MI-(10 kDaPHF-BA-(AF-HPA-Ala))) Conjugates

Example 1A, XMT-1535-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))); Example 2C,(10H1.11.4B)-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))); Example 3A,Rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) and AF-HPA or Example1C, XMT-1535-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))); Example 2C,(10H1.11.4B)-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))); Example 3B,Rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))); and AF-HPA wereevaluated for their antiproliferation properties in tumor cell lines invitro using Cell Titer-Glo (Promega Corp). OVCAR3 (ovarianadenocarcinoma cell line, not amplified, ATCC, Cat. # HTB-161) wascultured in RPMI medium with 20% FBS. TOV-21G (human ovarianadenocarcinoma cell line, not amplified, ATCC, Cat. # CRL-11730) wascultured in 1:1 mixture of MCDB 105 medium containing a finalconcentration of 1.5 g/L sodium bicarbonate and Medium 199 containing afinal concentration of 2.2 g/L sodium bicarbonate with 15% FBS. IGROV1(ovarian adenocarcinoma cell line, not amplified) was cultured in RPMImedium with 10% FBS. HCC-4006 (human lung carcinoma cell line, notamplified, ATCC, Cat. # ATCC® CRL-2871™) was cultured in in RPMI mediumwith 10% FBS.

For the cytotoxicity assay, cells were seeded at a density of 5000 cellsper well in 96 well plates and allowed to attach during overnightincubation at 37° C. in the presence of 5% CO₂. The media was thenreplaced with fresh media containing a range of the conjugates Examples1A, 2C, 3A or AF-HPA (100 nM to 0.1 pM) or Examples 1C, 2C, 3B or AF-HPA(100 nM to 0.1 pM) and the cells were incubated for 72 hours or 6 daysat 37° in the presence of 5% CO₂. Cell survival was measured usingCellTiter-Glo® Luminescent Cell Viability Assay (Promega, Madison, Wis.)as described in the kit instructions. Cell viability was normalized tountreated control and expressed as a percentage. The values were plottedand IC50 values calculated with GraphPad Prism software (San Diego,Calif.) using 4 parameter, variable slope, dose response curve fittingalgorithm. Table VA and VB give illustrative results for thecytotoxicity of the conjugates and AF-HPA.

TABLE VA NaPi2b Receptor AF- Tumor Cell number/ Example Example ExampleHPA¹ Line cell 1A (nM) 2C (nM) 3A (nM) (nM) OVCAR-3 ~32,000 0.002 0.0140.26 4.18 IGROV1 ~35,000 ~5.0 10.62 53.04 28.61 TOV-21G ~10,000 0.042.03 2.76 2.07 HCC-4006 ~52,000 0.13 2.96 1.83 0.68

TABLE VB NaPi2b Receptor AF- Tumor Cell number/ Example Example ExampleHPA¹ Line cell 1C (nM) 2C (nM) 3B (nM) (nM) OVCAR-3 ~32,000 0.01 0.111.13 1.34 IGROV1 ~35,000 0.38 2.73 37.3 25.94 TOV-21G ~10,000 6.68 41.5439.68 12.70 HCC-4006 ~52,000 0.35 3.17 5.29 7.83

1=payload equivalent

As shown in Tables VA and VB, the XMT-1535 antibody-polymer-drugconjugates are more potent than the (10H1.11.4B) antibody-polymer-drugconjugates or Rituximab antibody-polymer-drug conjugates in all thetested cell lines.

Example 6. Tumor Growth Response to Administration of NaPi2bAntibody-Polymer-Drug Conjugates

Female CB-17 SCID mice were subcutaneously implanted with OVCAR-3 (n=10for each group) or non-small cell lung cancertumor fragments (n=10 foreach group). Test compounds or vehicle were dosed IV as a single dose onday 1 or as indicated. Tumor size was measured at the times indicated inFIGS. 1 to 3 using digital calipers. Tumor volume was calculated and wasused to determine the delay in tumor growth. Mice were sacrificed whentumors reached a size of 1000 mm³. Tumor volumes are reported as themean±SEM for each group.

FIG. 1 provides the results for the tumor response in micesubcutaneously implanted with OVCAR-3 tumor fragments (n=10 for eachgroup) after IV administration of vehicle; XMT-1535; Example 3A,rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))); Example 4A((10H1.11.4B)-(MC-VC-PABA-MMAE)); Example 2C (10H1.11.4B)-(EG2-MI-(10kDa PHF-BA-(AF-HPA-Ala))) or Example 1B, XMT 1535-(EG2-MI-(10 kDaPHF-BA-(AF-HPA-Ala))), each at 3 mg/kg (0.21 mg/kg auristatin payloadequivalent dose) as a single dose at day 1. The vehicle, XMT-1535;conjugates Example 3A and Example 4A all showed an increase in tumorvolume with none of the treatments showing any partial regressions.Conjugates Example 2C and Example 1B each showed a decrease of tumorvolume. Conjugate Example 2C had a single partial regression response,while the conjugate Example 1B had 70% regressions consisting of 4partial regressions and 3 complete regressions. The median time toendpoint (TTE) was 57 days, corresponding to 118% tumor growth delay(TGD) (30.9 days). On Day 23 the tumor growth inhibition (TGI) outcomewas 95%. It was statistically different from control (P<0.001,Mann-Whitney test) and was above the 60% potential therapeutics activitythreshold.

FIG. 2 provides the results for the tumor response in micesubcutaneously implanted with OVCAR-3 tumor fragments (n=10 for eachgroup) after IV administration of vehicle; Example 3B,rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) at 3 mg/kg dosed weeklyfor 3 weeks; Example 4B ((10H1.11.4B)-(MC-VC-PABA-MMAE)) at 3 mg/kgdosed weekly for 3 weeks; Example 1C, XMT 1535-(EG2-MI-(10 kDaPHF-BA-(AF-HPA-Ala))) at 3 mg/kg as a single dose at day 1 or at 3 mg/kgdosed weekly for 3 weeks; or Example 1C, XMT 1535-(EG2-MI-(10 kDaPHF-BA-(AF-HPA-Ala))) at 5 mg/kg (0.36 mg/kg auristatin payloadequivalent dose) as a single dose at day 1. The vehicle and theconjugates Example 3B and Example 4B all showed an increase in tumorvolume with none of the treatments showing any partial regressions. Forthe conjugate Example 4B the tumor growth inhibition (TGI) at day 22differed statistically from controls (P<0.001, Mann-Whitney test) andwas at 63%—slightly above the 60% potential therapeutic activitythreshold. Conjugate Example 1B was most active in this study at alltreatment doses producing 80-100% regression responses. For thisconjugate the day 22 TGI outcome differed statistically from controls(P<0.001, Mann-Whitney test) and was at 96-97%.

FIG. 3 provides the results for the tumor response in patient derivedxenograft KRAS mutant non-small cell lung cancer model (CTG-0860) (n=10for each group) after IV administration of vehicle; Example 3B,rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))); Example 4B((10H1.11.4B)-(MC-VC-PABA-MMAE)); or Example 1C, XMT 1535-(EG2-MI-(10kDa PHF-BA-(AF-HPA-Ala))), each at 3 mg/kg dosed weekly for 3 weeks.Vehicle; conjugates Example 3B and Example 4B each showed an increase intumor volume. Comparison of Day 17 tumor volumes showed that tumorvolume of the Example 1C was significantly lower than that of theVehicle Control group (p<0.05) Analysis of data from Day 0 to Day 17showed that the Example 1C 3 mg/kg group had significantly lower tumorvolume compared to that of the vehicle control group (p ≤0.01). Therewere 2 PRs in the Example 1C 3 mg/kg group. There were no deaths in anyof the groups during the study.

FIG. 4 provides the results for the tumor response in patient derivedxenograft non-small cell lung cancer model (CTG-0852) (n=8 for eachgroup) after IV administration of vehicle; Example 3B,rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))); Example 4B((10H1.11.4B)-(MC-VC-PABA-MMAE)); Example 1C, XMT 1535-(EG2-MI-(10 kDaPHF-BA-(AF-HPA-Ala))), each at 3 mg/kg dosed weekly for 3 weeks orExample 1C, XMT 1535-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), at 5 mg/kgas a single dose on day 1. Analysis of data from Day 0 to Day 62 showedthat all of the groups had significantly lower tumor volume compared tothat of the vehicle control group (p<0.01 or p<0.0001). There were 3 PRsin the Example 4B 3 mg/kg group. There were 7 PRs in the Example 1C 5mg/kg group and 5 PRs, 1 CR, and 2 TFS in the Example 1C 3 mg/kg group.No PRs, CRs, or TFS in the Example 3B 3 mg/kg group. There were nodeaths in any of the groups during the study.

FIG. 5 provides the results for the tumor response in patient derivedxenograft non-small cell lung cancer model (ST1906, BRAF K601E mutation)(n=6 for each group) after IV administration of vehicle; and Example 1C,XMT 1535-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), at 3 mg/kg or 6 mg/kgdosed weekly for 3 weeks. Vehicle showed an increase in tumor volumewhile the conjugate Example 1C showed robust anti-tumor activity. Sixout of six exhibited TFS at day 60 after treatment with the conjugateExample 1C.

FIG. 7 provides the results for the tumor response in patient derivedxenograft non-small cell lung cancer model (CTG-0178) (n=8 for eachgroup) after IV administration of vehicle; Example 3B,rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))); Example 4B((10H1.11.4B)-(MC-VC-PABA-MMAE)); or Example 1C, XMT 1535-(EG2-MI-(10kDa PHF-BA-(AF-HPA-Ala))), each at 3 mg/kg dosed weekly for 3 weeks orExample 1C, XMT 1535-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) at 5 mg/kg asa single dose. Vehicle; conjugates Example 3B and Example 4B each showedan increase in tumor volume. The conjugate Example 1C at 3 mg/kg and 5mg/kg significantly lower tumor growth compared to that of the vehiclecontrol group (p<0.05). There were no deaths in any of the groups duringthe study.

FIG. 8 provides the results for the tumor response in patient derivedxenograft non-small cell lung cancer model (ST1437, EGFR wild type, Alkwild type mutation) (n=6 for each group) after IV administration ofvehicle; and Example 1C, XMT 1535-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))),at 3 mg/kg or 6 mg/kg dosed weekly for 3 weeks. Vehicle showed anincrease in tumor volume while the conjugate Example 1C showedanti-tumor activity. One out of six exhibited TFS at day 60 aftertreatment with the conjugate Example 1C.

FIG. 9 provides the results for the tumor response in patient derivedxenograft non-small cell lung cancer model (ST742, EMLK-Alktranslocation) (n=6 for each group) after IV administration of vehicle;and Example 1C, XMT 1535-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), at 3mg/kg or 6 mg/kg dosed weekly for 3 weeks. Vehicle showed an increase intumor volume while the conjugate Example 1C showed anti-tumor activity.Five out of six exhibited TFS at day 60 after treatment with theconjugate Example 1C.

FIG. 10 provides the results for the tumor response in patient derivedxenograft non-small cell lung cancer model (ST1243, EGFR amplification,KIT amplification, CDKN2A deletion) (n=6 for each group) after IVadministration of vehicle; and Example 1C, XMT 1535-(EG2-MI-(10 kDaPHF-BA-(AF-HPA-Ala))), at 3 mg/kg or 6 mg/kg dosed weekly for 3 weeks.Vehicle showed an increase in tumor volume while the conjugate Example1C showed anti-tumor activity.

Example 7: Cell Binding Affinity for XMT-1535 and NaPi2b-(EG2-MI-(10 kDaPHF-BA-(AF-HPA-Ala))) Conjugates Using Flow Cytometry

The cell surface binding of the Example 1C, XMT 1535-(EG2-MI-(10 kDaPHF-BA-(AF-HPA-Ala))) and anti-NaPi2B antibody XMT 1535, to antigenexpressing cells was evaluated by flow cytometry. TOV-21G or OVCAR3cells were grown overnight to approximately 90% confluent cultures inmedia, then were released from the plate surface by treatment withTrypsin-EDTA (Gibco-Thermo Fisher Scientific, USA). The detached cellswere washed once with ice cold media containing 6% goat serum andresuspended in the same media. 50,000 cells were aliquoted per well of aV-bottom, 96-well plate and incubated with a range of test articlesconcentrations (0.23-500 nM) in 100 μl Media (1:1 mixture MCDB105 (1.5g/L sodium bicarbonate) and Medium 199 (2.2 g/L sodium bicarbonate plus15% FBS)) with 6% goat serum on ice for 3 hours. The cells were thenwashed once with ice cold PBS and resuspended in 150 μl Media with 2%goat serum and 6 μg/ml of a secondary fluorescently labeled antibody,Alexa Fluor® 647-labelled goat anti-human IgG (Life Technologies, Cat#A-21445) for 1 hour on ice. The cells were washed once with ice cold PBSand suspended in 150 μl of ice cold PBS with 1% paraformaldehyde. Theamount of fluorescence bound per cell was determined by running 5000cells for each treatment on a MACSQuant flow cytometer (Miltenyi Biotec,Bergisch Gladbach, Germany). The median fluorescence value for eachtreatment was graphed, and the binding constant, K_(D) was calculatedfor each test article with GraphPad Prism software by non-linearregression using the one site specific binding model.

TABLE VI K_(D) (nM) K_(D) (nM) Test Compound TOV-21G OVCAR3 XMT 15352.46 6.59 Example 1C 3.97 3.84

As shown in Table VI, the naked antibody XMT-1535 and theXMT-1535-antibody-polymer-drug conjugate have similar binding affinitiesfor the tested cell line.

Example 8: Non-Human Primate Toxicological and PK Study

A single-dose toxicity study was conducted in cynomolgus monkeys withExample 1C, XMT-1535-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))). Theconjugate was administered to groups of 2 animals/sex at 0 mg/kg (Group1), 1.25 mg/kg (1074 μg/m² auristatin payload equivalents; Group 2), 2.5mg/kg (2147 μg/m² auristatin payload equivalents; Group 3) or 5 mg/kg(or 4294 μg/m² auristatin payload equivalents; Group 4) as a single ivinfusion. Blood was collected for hematology, coagulation, and serumchemistry analysis from all groups on days −7, 3, 8, 15, and 22. Bloodsamples were also collected at predose, 10 minutes, 1, 6, 24, 48, 72,96, and 168 hours following the end of the IV infusions, and on days 15and 22 for the PK determination.

The conjugate Example 1C was well tolerated in cynomolgus monkeys whenadministered up to 5 mg/kg (i.e., 4294 μg/m² auristatin payloadequivalents), with no observed target-mediated toxicity and limitedadverse findings. There was no evidence of bone marrow toxicity. Therewas no moribundity in Groups 2, 3, and 4. The test article relatedfindings are shown in Table VII below.

TABLE VII Terminal Necropsy Recovery Necropsy Organ 1.25 mg/kg 2.5 mg/kg5 mg/kg 1.25 mg/kg 2.5 mg/kg 5 mg/kg Bone Marrow None None None NoneNone None Liver* None None Minimal None None None hepatocyte apoptosis(1 female) Testes None None None None None None Lung None None Minimalmixed None None Minimal inflammatory cell mixed infiltrate (1 male)inflammatory cell infiltrate (1 male) Urinary None None Minimal mucosalNone None None Bladder apoptosis; occasional mitotic figures (1 male)Stomach Minimal None Mild focal None None None mucosal ulcerationneutrophil (1 male) infiltrate (1 female) Cecum None Mild focal NoneNone None None ulceration (1 female) *Minimal Kupffer cell hypertrophywith occasional mitotic figures was seen in all test article treatedanimals at terminal and recovery necropsies (non-adverse finding).

One animal in all groups was affected by adverse changes involving thegastrointestinal tract at terminal necropsy. Recovery animals had testarticle related findings (one in each tissue) in the liver (Groups 2 to4), spleen (Groups 3 and 4), and lung (Group 4) but none of these wereconsidered adverse. There was transient increase in neutrophil countsand a monocytosis in conjunction with increased globulin concentrations,decreased albumin levels and decreased albumin to globulin ratios. Also,elevated creatine kinase (CK) and aspartate aminotransferase (AST)levels were observed. None of the alterations detected persisted throughthe recovery period. There was no myelosuppression and no neutropenia.In addition, there were no test article gross findings at necropsy orrelated body weight loss. Based on the efficacy and this non-humanprimate toxicological study therapeutic index for the conjugate Example1C, XMT-1535-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) is about 6.

The blood samples were analyzed by LC-MS/MS to determine theconcentration of total AF-HPA and by ELISA to determine the totalantibody concentration at each time point. FIGS. 6A and 6B show theplasma pharmacokinetics for the total antibody and total drugrespectively after administration of a single dose in cynomolgus monkeyswith the conjugate Example 1C at 1.25 mg/kg (1074 μg/m² auristatinpayload equivalents; Group 2), 2.5 mg/kg (2147 μg/m² auristatin payloadequivalents; Group 3) or 5 mg/kg (or 4294 μg/m² auristatin payloadequivalents; Group 4). Table VIII gives the calculatedAUC_(0 to 504 hrs) and half-lives for total antibody and the totalAF-HPA. Free drugs (i.e., unconjugated AF and unconjugated AF-HPA) werenot detected (LLOQ of the LC MS/MS method was 1 ng/mL).

TABLE VIII Plasma Exposure AUC_(0-504 hrs) Dose Normalized PlasmaHalf-life T_(1/2), (μg · hrs/mL/mg/kg) Dose Range Average (Days) TotalAntibody Total Drug Total Antibody Total Drug Drug Release 1,407 ± 99157 ± 13 8.8 ± 0.7 5.2 ± 0.2 11.6 ± 0.9

The XMT-1535-polymer-drug conjugate had a half-life of ˜9 days with anAUC_(0-504 hrs) of ˜1.4 mg.hr/mL/mg/kg. The total AF-HPA had a half-lifeof ˜5 days with an AUC_(0-504 hrs) of ˜157 μg.hr/mL/mg/kg. The conjugatedemonstrated good stability in plasma with little or no detection offree drugs.

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

1. A conjugate comprising an isolated antibody that specifically bindsto the extracellular region of SLC34A2 and one or more D-carryingpolymeric scaffolds connected to the isolated antibody, wherein each ofthe one or more D-carrying polymeric scaffolds independently is ofFormula (Ic):

wherein: the scaffold comprises poly(1-hydroxymethylethylenehydroxymethyl-formal) (PHF) having a molecular weight ranging from about2 kDa to about 40 kDa; each occurrence of D is independently atherapeutic agent having a molecular weight of ≤5 kDa; L^(D1) is acarbonyl-containing moiety; each occurrence of

is independently a first linker that contains a biodegradable bond sothat when the bond is broken, D is released in an active form for itsintended therapeutic effect; and the

between L^(D1) and D denotes direct or indirect attachment of D toL^(D1); each occurrence of

is independently a second linker not yet connected to the isolatedantibody, in which L^(P2) is a moiety containing a functional group thatis yet to form a covalent bond with a functional group of the isolatedantibody, and the

between L^(D1) and L^(P2) denotes direct or indirect attachment ofL^(P2) to L^(D1), and each occurrence of the second linker is distinctfrom each occurrence of the first linker; each occurrence of

is independently a third linker that connects each D-carrying polymericscaffold to the isolated antibody, in which the terminal

attached to L^(P2) denotes direct or indirect attachment of L^(P2) tothe isolated antibody upon formation of a covalent bond between afunctional group of L^(P2) and a functional group of the isolatedantibody; and each occurrence of the third linker is distinct from eachoccurrence of the first linker; m is an integer from 1 to about 300, m₁is an integer from 1 to about 140, m₂ is an integer from 1 to about 40,m₃ is an integer from 0 to about 18, m₄ is an integer from 1 to about10; the sum of m, m₁, m₂, m₃, and m₄ ranges from 15 to 300; and thetotal number of L^(P2) connected to the isolated antibody is 10 or less.2. The conjugate of claim 1, wherein the isolated antibody thatspecifically binds the extracellular region of SLC34A2 comprises avariable heavy chain complementarity determining region 1 (CDRH1)comprising the amino acid sequence GYTFTGYNIH (SEQ ID NO: 5), a variableheavy chain complementarity determining region 2 (CDRH2) comprising theamino acid sequence AIYPGNGDTSYKQKFRG (SEQ ID NO: 6), a variable heavychain complementarity determining region 3 (CDRH3) comprising the aminoacid sequence GETARATFAY (SEQ ID NO: 7), a variable light chaincomplementarity determining region 1 (CDRL1) comprising the amino acidsequence SASQDIGNFLN (SEQ ID NO: 8), a variable light chaincomplementarity determining region 2 (CDRL2) comprising the amino acidsequence YTSSLYS (SEQ ID NO: 9), a variable light chain complementaritydetermining region 3 (CDRL3) comprising the amino acid sequenceQQYSKLPLT (SEQ ID NO: 10).
 3. The conjugate of claim 1, wherein theisolated antibody that specifically binds the extracellular region ofSLC34A2 comprises a heavy chain variable sequence comprising the aminoacid sequence of SEQ ID NO: 3 or an amino acid sequence at least 90%identical thereto and a light chain variable sequence comprising theamino acid sequence of SEQ ID NO: 4 or an amino acid sequence at least90% identical thereto.
 4. The conjugate of claim 1, wherein the isolatedantibody that specifically binds the extracellular region of SLC34A2comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:1 or an amino acid sequence at least 90% identical thereto and a lightchain comprising the amino acid sequence of SEQ ID NO: 2 or an aminoacid sequence at least 90% identical thereto.
 5. The conjugate of claim1, wherein the isolated antibody that specifically binds theextracellular region of SLC34A2 is a monoclonal antibody.
 6. Theconjugate of claim 1, wherein the isolated antibody that specificallybinds the extracellular region of SLC34A2 is a rabbit, mouse, chimeric,humanized or fully human monoclonal antibody.
 7. The conjugate of claim1, wherein the isolated antibody that specifically binds theextracellular region of SLC34A2 is an IgG isotype.
 8. The conjugate ofclaim 1, wherein the isolated antibody that specifically binds theextracellular region of SLC34A2 is an IgG1 isotype.
 9. The conjugate ofclaim 1, wherein the isolated antibody (i) competes for specific bindingto human NaPi2b with an isolated antibody comprising a variable heavychain complementarity determining region 1 (CDRH1) comprising the aminoacid sequence GYTFTGYNIH (SEQ ID NO: 5), a variable heavy chaincomplementarity determining region 2 (CDRH2) comprising the amino acidsequence AIYPGNGDTSYKQKFRG (SEQ ID NO: 6), a variable heavy chaincomplementarity determining region 3 (CDRH3) comprising the amino acidsequence GETARATFAY (SEQ ID NO: 7), a variable light chaincomplementarity determining region 1 (CDRL1) comprising the amino acidsequence SASQDIGNFLN (SEQ ID NO: 8), a variable light chaincomplementarity determining region 2 (CDRL2) comprising the amino acidsequence YTSSLYS (SEQ ID NO: 9), a variable light chain complementaritydetermining region 3 (CDRL3) comprising the amino acid sequenceQQYSKLPLT (SEQ ID NO: 10), or (ii) competes for specific binding tohuman NaPi2b with an isolated antibody comprising a heavy chain variablesequence comprising the amino acid sequence of SEQ ID NO: 3 or an aminoacid sequence at least 90% identical thereto and a light chain variablesequence comprising the amino acid sequence of SEQ ID NO: 4 or an aminoacid sequence at least 90% identical thereto, or with an isolatedantibody comprising a heavy chain comprising the amino acid sequence ofSEQ ID NO: 1 or an amino acid sequence at least 90% identical theretoand a light chain comprising the amino acid sequence of SEQ ID NO: 2 oran amino acid sequence at least 90% identical thereto.
 10. (canceled)11. The conjugate of claim 1, wherein the sum of m, m₁, m₂, m₃ and m₄ranges from 15 to 150, m₁ is an integer from 1 to 70, m₂ is an integerfrom 1 to 20, m₃ is an integer from 0 to 10, and PHF has a molecularweight ranging from about 2 kDa to about 20 kDa.
 12. The conjugate ofclaim 1, wherein the sum of m, m₁, m₂, m₃ and m₄ ranges from 20 to 110,m₁ is an integer from 2 to 50, m₂ is an integer from 2 to 15, m₃ is aninteger from 0 to 8; and PHF has a molecular weight ranging from about 3kDa to about 15 kDa.
 13. The conjugate of claim 1, wherein the sum of m,m₁, m₂, m₃ and m₄ ranges from 40 to 75, m₁ is an integer from 2 to 35,m₂ is an integer from 2 to 10, m₃ is an integer from 0 to 5; and PHF hasa molecular weight ranging from about 5 kDa to about 10 kDa.
 14. Theconjugate of claim 1, wherein the functional group of L^(P2) is selectedfrom —SR^(p), —S—S-LG,

and halo, in which LG is a leaving group, R^(p) is H or a sulfurprotecting group, and one of X_(a) and X_(b) is H and the other is awater-soluble maleimido blocking moiety, or X_(a) and X_(b), togetherwith the carbon atoms to which they are attached for a carbon-carbondouble bond.
 15. The conjugate of claim 1, wherein L^(D1) comprises—X—(CH₂)_(v)—C(═O)— with X directly connected to the carbonyl group of

in which X is CH₂, O, or NH, and v is an integer from 1 to
 6. 16. Theconjugate of claim 1, wherein each occurrence of

is independently—C(═O)—X—(CH₂)_(v)—C(═O)—NH—(CH₂)_(u)—NHC(═O)—(CH₂)_(w)—(OCH₂CH₂)_(x)—NHC(═O)—(CH₂)_(y)-M,in which X is CH₂, O, or NH, each of v, u, w, x and y independently isan integer from 1 to 6, and M is

wherein one of X_(a) and X_(b) is H and the other is a water-solublemaleimido blocking moiety, or X_(a) and X_(b), together with the carbonatoms to which they are attached for a carbon-carbon double bond. 17.The conjugate of claim 16, wherein each of v, u, w, x and y is
 2. 18.The conjugate of claim 1, wherein each occurrence of D independently isselected from vinca alkaloids, auristatins, tubulysins, duocarmycins,calicheamicins, topoisomerase I inhibitors, PI3 kinase inhibitors, MEKinhibitors, KSP inhibitors, pyrrolobenzodiazepines, non-naturalcamptothecins, maytansinoids, DNA-binding drugs, DNA-alkylating drugs,RNA polymerase inhibitors, and analogs thereof.
 19. The conjugate ofclaim 1, wherein each of the one or more D-carrying polymeric scaffoldsindependently is of Formula (Id):

wherein: m_(3a) is an integer from 0 to about 17, m_(3b) is an integerfrom 1 to about 8, and the terminal

denotes the direct attachment of the one or more polymeric scaffolds tothe isolated antibody.
 20. The conjugate of claim 1, wherein each of theone or more D-carrying polymeric scaffolds independently is of Formula(If):

wherein: m is an integer from 1 to about 300, m₁ is an integer from 1 toabout 140, m₂ is an integer from 1 to about 40, m_(3a) is an integerfrom 0 to about 17, m_(3b) is an integer from 1 to about 8; the sum ofm_(3a) and m_(3b) ranges from 1 and about 18; and the sum of m, m₁, m₂,m_(3a), and m_(3b) ranges from 15 to about 300; the terminal

denotes the attachment of one or more polymeric scaffolds to theisolated antibody that specifically binds to SLC34A2, wherein theisolated antibody that specifically binds to SLC34A2 is an isolatedantibody that comprises a variable light chain complementaritydetermining region 1 (CDRL1) comprising the amino acid sequenceSASQDIGNFLN (SEQ ID NO: 8); a variable light chain complementaritydetermining region 2 (CDRL2) comprising the amino acid sequence YTSSLYS(SEQ ID NO: 9); a variable light chain complementarity determiningregion 3 (CDRL3) comprising the amino acid sequence QQYSKLPLT (SEQ IDNO: 10); a variable heavy chain complementarity determining region 1(CDRH1) comprising the amino acid sequence GYTFTGYNIH (SEQ ID NO: 5); avariable heavy chain complementarity determining region 2 (CDRH2)comprising the amino acid sequence AIYPGNGDTSYKQKFRG (SEQ ID NO: 6); anda variable heavy chain complementarity determining region 3 (CDRH3)comprising the amino acid sequence GETARATFAY (SEQ ID NO: 7); and theratio between the PHF and the antibody is 10 or less.
 21. The conjugateof claim 20, wherein the PHF in Formula (If) has a molecular weightranging from about 2 kDa to about 20 kDa, the sum of m, m₁, m₂, m_(3a)and m_(3b) ranges from about 15 to about 150, m₁ is an integer from 1 toabout 70, m₂ is an integer from 1 to about 20, m_(3a) is an integer from0 to about 9, m_(3b) is an integer from 1 to about 8, the sum of m_(3a)and m_(3b) ranges from 1 and about 10, and the ratio between the PHF andthe isolated antibody that specifically binds to SLC34A2 is an integerfrom 2 to about
 8. 22. The conjugate of claim 20, wherein the PHF inFormula (If) has a molecular weight ranging from about 3 kDa to about 15kDa, the sum of m, m₁, m₂, m_(3a) and m_(3b) ranges from about 20 toabout 110, m₁ is an integer from 2 to about 50, m₂ is an integer from 2to about 15, m_(3a) is an integer from 0 to about 7, m_(3b) is aninteger from 1 to about 8, the sum of m_(3a) and m_(3b) ranges from 1and about 8, and the ratio between the PHF and the isolated antibodythat specifically binds to SLC34A2 is an integer from 2 to about
 8. 23.The conjugate of claim 20, wherein the PHF in Formula (If) has amolecular weight ranging from about 5 kDa to about 10 kDa, the sum of m,m₁, m₂, m_(3a) and m_(3b) ranges from about 40 to about 75, m₁ is aninteger from about 2 to about 35, m₂ is an integer from about 2 to about10, m_(3a) is an integer from 0 to about 4, m_(3b) is an integer from 1to about 5, the sum of m_(3a) and m_(3b) ranges from 1 and about 5, andthe ratio between the PHF and the isolated anti-antibody thatspecifically binds to SLC34A2 is an integer from 2 to about
 8. 24. Theconjugate of claim 20, wherein the PHF in Formula (If) has a molecularweight ranging from about 5 kDa to about 10 kDa, the sum of m, m₁, m₂,m_(3a) and m_(3b) ranges from about 40 to about 75, m₁ is an integerfrom about 2 to about 35, m₂ is an integer from about 2 to about 10,m_(3a) is an integer from 0 to about 4, m_(3b) is an integer from 1 toabout 5, the sum of m_(3a) and m_(3b) ranges from 1 and about 5, and theratio between the PHF and the isolated antibody that specifically bindsto SLC34A2 is an integer from 2 to about
 6. 25. A pharmaceuticalcomposition comprising a conjugate of claim 20 and a pharmaceuticallyacceptable carrier.
 26. A method of preparing a conjugate according toclaim 1, comprising reacting an isolated antibody that specificallybinds to SLC34A2 with a D-carrying polymeric scaffold of Formula (Ia)such that the conjugate is formed:

wherein: L^(D1) is a carbonyl-containing moiety; each occurrence of

is independently a first linker that contains a biodegradable bond sothat when the bond is broken, D is released in an active form for itsintended therapeutic effect; and the

between L^(D1) and D denotes direct or indirect attachment of D toL^(D1); each occurrence of

is independently a second linker not yet connected to the isolatedantibody, in which L² is a moiety containing a functional group that isyet to form a covalent bond with a functional group of the isolatedantibody, and the

between L^(D1) and L^(P2) denotes direct or indirect attachment ofL^(P2) to L^(D1), and each occurrence of the second linker is distinctfrom each occurrence of the first linker; m is an integer from 1 toabout 300, m₁ is an integer from 1 to about 140, m₂ is an integer from 1to about 40, m₃ is an integer from 1 to about 18, and the sum of m, m₁,m₂ and m₃ ranges from 15 to about
 300. 27. A method of alleviating asymptom of a cancer in a subject in need thereof, the method comprisingadministering a conjugate according to claim 20 to the subject in anamount sufficient to alleviate the symptom of the cancer.
 28. The methodof claim 27, wherein the subject is human.
 29. The method of claim 27,wherein the cancer is selected from the group consisting of ovariancancer, thyroid cancer, colorectal cancer, lung cancer, non-small celllung cancer (NSCLC), breast cancer, kidney cancer and salivary ductcarcinoma.
 30. The method of claim 27, wherein the cancer is selectedfrom the group consisting of non-small cell lung cancer (NSCLC) andovarian cancer.
 31. The method of claim 30, wherein the non-small celllung cancer is non-squamous non-small cell lung cancer.
 32. The methodof claim 30, wherein the ovarian cancer is epithelial ovarian cancer.33. The method of claim 27, further comprising administration of atherapeutic agent to the subject.
 34. The method of claim 27, whereinthe subject has one or more ovarian cancers selected from recurrentovarian cancer, platinum-sensitive ovarian cancer, platinum-refractoryovarian cancer, and platinum-resistant ovarian cancer; or the subjecthas advanced ovarian cancer and has not received any prior chemotherapyfor treating cancer.
 35. (canceled)